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Increasing evidence has shown that voltage-gated L-type Ca

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., 脳の神経活動を可視化する新規マウス系統を開発 --高感度・高速カルシウムセンサーによる神経活動の計測に成功--. 京都大学プレスリリース. 2022-02-15.

In patients born blind with retinal dystrophies, understanding the critical periods of cortical plasticity is important for successful visual restoration. In this study, we sought to model childhood blindness and investigate the plasticity of visual pathways. To this end, we generated double-mutant (Pde6c(cpfl1/cpfl1)Gnat1(IRD2/IRD2)) mice with absent rod and cone photoreceptor function, and we evaluated their response for restoring rod (GNAT1) function through gene therapy. Despite the limited effectiveness of gene therapy in restoring visual acuity in patients with retinal dystrophy, visual acuity was, unexpectedly, successfully restored in the mice at the level of the primary visual cortex in this study. This success in visual restoration, defined by changes in the quantified optokinetic response and pattern visually evoked potential, was achieved regardless of the age at treatment (up to 16 months). In the contralateral visual cortex, cortical plasticity, tagged with light-triggered transcription of Arc, was also restored after the treatment in blind mice carrying an Arc promoter-driven reporter gene, dVenus. Our results demonstrate the remarkable plasticity of visual circuits for one of the two photoreceptor mechanisms in older as well as younger mice with congenital blindness due to retinal dystrophies.

Despite many association studies linking gene polymorphisms and mutations of L-type voltage-gated Ca(2+) channels (VGCCs) in neurodevelopmental disorders such as autism and schizophrenia, the roles of specific L-type VGCC during brain development remain unclear. Calcium signaling has been shown to be essential for neurodevelopmental processes such as sculpting of neurites, functional wiring, and fine tuning of growing networks. To investigate this relationship, we performed submembraneous calcium imaging using a membrane-tethered genetically encoded calcium indicator (GECI) Lck-G-CaMP7. We successfully recorded spontaneous regenerative calcium transients (SRCaTs) in developing mouse excitatory cortical neurons prepared from both sexes before synapse formation. SRCaTs originated locally in immature neurites independently of somatic calcium rises and were significantly more elevated in the axons than in dendrites. SRCaTs were not blocked by tetrodoxin, a Na(+) channel blocker, but were strongly inhibited by hyperpolarization, suggesting a voltage-dependent source. Pharmacological and genetic manipulations revealed the critical importance of the Ca(v)1.2 (CACNA1C) pore-forming subunit of L-type VGCCs, which were indeed expressed in immature mouse brains. Consistently, knocking out Ca(v)1.2 resulted in significant alterations of neurite outgrowth. Furthermore, expression of a gain-of-function Ca(v)1.2 mutant found in Timothy syndrome, an autosomal dominant multisystem disorder exhibiting syndromic autism, resulted in impaired radial migration of layer 2/3 excitatory neurons, whereas postnatal abrogation of Ca(v)1.2 enhancement could rescue cortical malformation. Together, these lines of evidence suggest a critical role for spontaneous opening of L-type VGCCs in neural development and corticogenesis and indicate that L-type VGCCs might constitute a perinatal therapeutic target for neuropsychiatric calciochannelopathies. SIGNIFICANCE STATEMENT Despite many association studies linking gene polymorphisms and mutations of L-type voltage-gated Ca(2+) channels (VGCCs) in neurodevelopmental disorders such as autism and schizophrenia, the roles of specific L-type VGCCs during brain development remain unclear. We here combined the latest Ca(2+) indicator technology, quantitative pharmacology, and in utero electroporation and found a hitherto unsuspected role for L-type VGCCs in determining the Ca(2+) signaling landscape of mouse immature neurons. We found that malfunctional L-type VGCCs in immature neurons before birth might cause errors in neuritic growth and cortical migration. Interestingly, the retarded corticogenesis phenotype was rescued by postnatal correction of L-type VGCC signal aberration. These findings suggest that L-type VGCCs might constitute a perinatal therapeutic target for neurodevelopment-associated psychiatric disorders.

Summary 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 Download : Download video (56MB)

Neuronal migration is a crucial event in neuronal development for the construction of brain architecture and neuronal networks. Newborn neurons proliferate in the germinal zone and start migration toward their final destination. Migrating neurons adopt different routes, cell shapes and migratory modes depending on extracellular factors and outer physical substrates. Intracellular Ca2+ is an essential second messenger that regulates diverse cellular functions by activating Ca2+-dependent signalling molecules that underlie Ca2+-responsive cellular functions. Neuronal migration during brain architecture construction is no exception. Spontaneous Ca2+ transients are observed in several types of migrating neurons, and a series of Ca2+-dependent signalling molecules governing neuronal migration has been identified. In this review, we first summarize the molecular mechanisms that trigger intracellular Ca2+ elevation in migrating neurons. In the latter half of this review, we provide an overview of the literature on Ca2+-dependent signalling molecules underlying neuronal migration.

Fluorescent Ca(2+) reporters are widely used as readouts of neuronal activities. Here we designed R-CaMP2, a high-affinity red genetically encoded calcium indicator (GECI) with a Hill coefficient near 1. Use of the calmodulin-binding sequence of CaMKK-α and CaMKK-β in lieu of an M13 sequence resulted in threefold faster rise and decay times of Ca(2+) transients than R-CaMP1.07. These features allowed resolving single action potentials (APs) and recording fast AP trains up to 20-40 Hz in cortical slices. Somatic and synaptic activities of a cortical neuronal ensemble in vivo were imaged with similar efficacy as with previously reported sensitive green GECIs. Combining green and red GECIs, we successfully achieved dual-color monitoring of neuronal activities of distinct cell types, both in the mouse cortex and in freely moving Caenorhabditis elegans. Dual imaging using R-CaMP2 and green GECIs provides a powerful means to interrogate orthogonal and hierarchical neuronal ensembles in vivo.

SummaryThe Arc/Arg3.1 gene product is rapidly upregulated by strong synaptic activity and critically contributes to weakening synapses by promoting AMPA-R endocytosis. However, how activity-induced Arc is redistributed and determines the synapses to be weakened remains unclear. Here, we show targeting of Arc to inactive synapses via a high-affinity interaction with CaMKIIβ that is not bound to calmodulin. Synaptic Arc accumulates in inactive synapses that previously experienced strong activation and correlates with removal of surface GluA1 from individual synapses. A lack of CaMKIIβ either in vitro or in vivo resulted in loss of Arc upregulation in the silenced synapses. The discovery of Arc's role in “inverse” synaptic tagging that is specific for weaker synapses and prevents undesired enhancement of weak synapses in potentiated neurons reconciles essential roles of Arc both for the late phase of long-term plasticity and for reduction of surface AMPA-Rs in stimulated neurons.

Ca(2+) -regulated reorganization of actin cytoskeleton is one of the key cell biological events that critically regulate neuronal morphogenesis during circuit formation, spinogenesis during synapse development, and activity-dependent structural plasticity at mature synapses. However, it remains unclear as to what extent the underlying Ca(2+) signaling processes are shared or segregated. Here, we present evidence from the literature that collectively begins to suggest that distinct calmodulin-dependent protein kinase (CaMK) isoforms are differentially expressed in time and in subcellular space, and thus may be selectively activated and engaged by distinct upstream stimuli; each CaMK isoform, in turn, couples to related, but separate, cytoskeletal and transcriptional regulatory pathways, dependent on its abundance or physical proximity with either the upstream or downstream signaling complexes. These signal transduction characteristics provide the basis for better understanding the role of excitation-morphogenesis coupling via multiple CaMKs during neuronal circuit and synapse formation.

Ca2+signaling plays important roles during both axonal and dendritic growth. Yet whether and how Ca2+rises may trigger and contribute to the development of long-range cortical connections remains mostly unknown. Here, we demonstrate that two separate limbs of the Ca2+/calmodulin-dependent protein kinase kinase (CaMKK)–CaMKI cascades, CaMKK–CaMKIα and CaMKK–CaMKIγ, critically coordinate axonal and dendritic morphogenesis of cortical neurons, respectively. The axon-specific morphological phenotype required a diffuse cytoplasmic localization and a strikingly α-isoform-specific kinase activity of CaMKI. Unexpectedly, treatment with muscimol, a GABAAreceptor agonist, selectively stimulated elongation of axons but not of dendrites, and the CaMKK–CaMKIα cascade critically mediated this axonogenic effect. Consistent with these findings, during early brain development,in vivoknockdown of CaMKIα significantly impaired the terminal axonal extension and thereby perturbed the refinement of the interhemispheric callosal projections into the contralateral cortices. Our findings thus indicate a novel role for the GABA-driven CaMKK–CaMKIα cascade as a mechanism critical for accurate cortical axon pathfinding, an essential process that may contribute to fine-tuning the formation of interhemispheric connectivity during the perinatal development of the CNS.

The neuronal immediate early gene Arc / Arg-3.1 is widely used as one of the most reliable molecular markers for intense synaptic activity in vivo. However, the cis -acting elements responsible for such stringent activity dependence have not been firmly identified. Here we combined luciferase reporter assays in cultured cortical neurons and comparative genome mapping to identify the critical synaptic activity-responsive elements (SARE) of the Arc / Arg-3.1 gene. A major SARE was found as a unique ≈100-bp element located at >5 kb upstream of the Arc / Arg-3.1 transcription initiation site in the mouse genome. This single element, when positioned immediately upstream of a minimal promoter, was necessary and sufficient to replicate crucial properties of endogenous Arc / Arg-3.1' s transcriptional regulation, including rapid onset of transcription triggered by synaptic activity and low basal expression during synaptic inactivity. We identified the major determinants of SARE as a unique cluster of neuronal activity-dependent cis -regulatory elements consisting of closely localized binding sites for CREB, MEF2, and SRF. Consistently, a SARE reporter could readily trace and mark an ensemble of cells that have experienced intense activity in the recent past in vivo. Taken together, our work uncovers a novel transcriptional mechanism by which a critical 100-bp element, SARE, mediates a predominant component of the synapse-to-nucleus signaling in ensembles of Arc/Arg-3.1-positive activated neurons.

Despite the critical importance of Ca(2+)/calmodulin (CaM)-dependent protein kinase (CaMK) II signaling in neuroplasticity, only a limited amount of work has so far been available regarding the presence and significance of another predominant CaMK subfamily, the CaMKI/CaMKIV family, in the central nervous system. We here searched for kinases with a core catalytic structure similar to CaMKI and CaMKIV. We isolated full-length cDNAs encoding three mouse CaMKI/CaMKIV-related kinases, CLICK-I (CL1)/doublecortin and CaM kinase-Like (DCAMKL)1, CLICK-II (CL2)/DCAMKL2, and CLICK-I,II-related (CLr)/DCAMKL3, the kinase domains of which had an intermediate homology not only to CaMKI/CaMKIV but also to CaMKII. Furthermore, CL1, CL2, and CLr were highly expressed in the central nervous system, in a neuron-specific fashion. CL1alpha and CL1beta were shorter isoforms of DCAMKL1, which lacked the doublecortin-like domain (Dx). In contrast, CL2alpha and CL2beta contained a full N-terminal Dx, whereas CLr only possessed a partial and dysfunctional Dx. Interestingly, despite a large similarity in the kinase domain, CL1/CL2/CLr had an impact on CRE-dependent gene expression distinct from that of the related CaMKI/CaMKIV and CaMKII. Although these were previously shown to activate Ca(2+)/cAMP-response element-binding protein (CREB)-dependent transcription, we here show that CL1 and CL2 were unable to significantly phosphorylate CREB Ser-133 and rather inhibited CRE-dependent gene expression by a dominant mechanism that bypassed CREB and was mediated by phosphorylated TORC2.

CREB is a transcription factor critical for long-term synaptic plasticity. Intriguingly, recent work has elucidated a role for CREB, as well as upstream CREB kinases, in the control of activity-dependent neuronal survival. Additionally, analysis of the molecular pathology of polyglutamine-repeat diseases suggest that alteration of pCREB-CBP function may underlie, at least in part, the neurodegenerative process. Taken together, these new findings support the idea that Ca(2+)/CREB/CBP-dependent gene regulation might be a shared mechanism critical in both long-term synaptic plasticity and neuronal survival.

During a screen for novel putative Ca(2+)/calmodulin-dependent protein kinase (CaMK)-like CREB kinases (CLICKs), we have cloned a full-length cDNA for CLICK-III/CaMKIgamma, an isoform of the CaMKI family with an extended C-terminal domain ending with CAAX motif (where AA is aliphatic acid). As expected from the similarity of its kinase domain with the other CaMKI isoforms, full activation of CLICK-III/CaMKIgamma required both Ca(2+)/CaM and phosphorylation by CaMKK. We also found that Ca(2+)/cAMP-response element-binding protein (CREB) was a good substrate for CLICK-III/CaMKIgamma, at least in vitro. Interestingly enough, CLICK-III/CaMKIgamma transcripts were most abundant in neurons, with the highest levels in limited nuclei such as the central nucleus of the amygdala (CeA) and the ventromedial hypothalamus. Consistent with the presence of the CAAX motif, CLICK-III/CaMKIgamma was found to be anchored to various membrane compartments, especially to Golgi and plasma membranes. Both point mutation in the CAAX motif and treatment with compactin, a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, disrupted such membrane localization, suggesting that membrane localization of CLICK-III/CaMKIgamma occurred in a prenylation-dependent way. These findings provide a novel mechanism by which neuronal CaMK activity could be targeted to specific membrane compartments.

Rho-GTPase has been implicated in axon outgrowth. However, not all of the critical steps controlled by Rho have been well characterized. Using cultured cerebellar granule neurons, we show here that stromal cell-derived factor (SDF)-1alpha, a neural chemokine, is a physiological ligand that can turn on two distinct Rho-dependent pathways with opposite consequences. A low concentration of the ligand stimulated a Rho-dependent pathway that mediated facilitation of axon elongation. In contrast, Rho/ROCK activation achieved by a higher concentration of SDF-1alpha caused repression of axon formation and induced no more increase in axon length. However, even at this higher concentration a Rho-dependent axon elongating activity could be recovered upon removal of ROCK activity using Y-27632. SDF-1alpha-induced axon elongating activity under ROCK inhibition was replicated by the dominant-active form of the mammalian homologue of the Drosophila gene Diaphanous (mDia)1 and counteracted by its dominant-negative form. Furthermore, RNAi knockdown of mDia1 abolished SDF-1alpha-induced axon elongation. Together, our results support a critical role for an SDF-1alpha/Rho/mDia1 pathway in mediating axon elongation.

In the field of molecular and cellular neuroscience, it is not a trivial task to see the forest for the trees, where numerous, and seemingly independent, molecules often work in concert to control critical steps of synaptic plasticity and signalling. Here, we will first summarize our current knowledge on essential activity-dependent transcription factors (TFs) such as CREB, MEF2, Npas4 and SRF, then examine how various transcription cofactors (TcoFs) also contribute to defining the transcriptional outputs during learning and memory. This review finally attempts a provisory synthesis that sheds new light on some of the emerging principles of neuronal circuit dynamics driven by activity-regulated gene transcription to help better understand the intricate relationship between activity-dependent gene expression and cognitive behavior.

Identifying the neuronal ensembles that respond to specific stimuli and mapping their projection patterns in living animals are fundamental challenges in neuroscience. To this end, we engineered a synthetic promoter, the enhanced synaptic activity-responsive element (E-SARE), that drives neuronal activity-dependent gene expression more potently than other existing immediate-early gene promoters. Expression of a drug-inducible Cre recombinase downstream of E-SARE enabled imaging of neuronal populations that respond to monocular visual stimulation and tracking of their long-distance thalamocortical projections in living mice. Targeted cell-attached recordings and calcium imaging of neurons in sensory cortices revealed that E-SARE reporter expression correlates with sensory-evoked neuronal activity at the single-cell level and is highly specific to the type of stimuli presented to the animals. This activity-dependent promoter can expand the repertoire of genetic approaches for high-resolution anatomical and functional analysis of neural circuits.

Ca(2+) -regulated reorganization of actin cytoskeleton is one of the key cell biological events that critically regulate neuronal morphogenesis during circuit formation, spinogenesis during synapse development, and activity-dependent structural plasticity at mature synapses. However, it remains unclear as to what extent the underlying Ca(2+) signaling processes are shared or segregated. Here, we present evidence from the literature that collectively begins to suggest that distinct calmodulin-dependent protein kinase (CaMK) isoforms are differentially expressed in time and in subcellular space, and thus may be selectively activated and engaged by distinct upstream stimuli; each CaMK isoform, in turn, couples to related, but separate, cytoskeletal and transcriptional regulatory pathways, dependent on its abundance or physical proximity with either the upstream or downstream signaling complexes. These signal transduction characteristics provide the basis for better understanding the role of excitation-morphogenesis coupling via multiple CaMKs during neuronal circuit and synapse formation.

A large number of molecular mechanisms contribute to ensuring that the neuronal transcriptome can be adapted in function of the various kinds of external and internal events that the neuronal network is exposed to. In recent years, activity-induced gene expression/ protein synthesis has received much attention as a potential mechanism likely to play a significant role in synaptic plasticity and long-term memory formation. The involved regulatory processes are intrinsically complex, and we still lack a detailed understanding of how specific neuronal nuclear factors are activated and modulated in concert to give rise to reliable and reproducible gene induction. In this chapter, we will consider the regulation of one of the most studied neuronal nuclear factor, the transcription factor CREB (Ca2+/cAMP-response element-binding protein).

Calcium (Ca2+) signaling is essential for a variety of cellular responses and higher biological functions. Ca2+/calmodulin-dependent kinases (CaMKs) and the phosphatase calcineurin activate distinct downstream pathways that are mediated by the transcription factors cAMP response element (CRE)-binding protein (CREB) and nuclear factor of activated T cells (NFAT), respectively1. The importance of the calcineurin-NFAT pathway in bone metabolism has been demonstrated in osteoclasts, osteoblasts and chondrocytes2,3,4,5. However, the contribution of the CaMK-CREB pathway is poorly understood, partly because of the difficulty of dissecting the functions of homologous family members6,7,8. Here we show that the CaMKIV-CREB pathway is crucial for osteoclast differentiation and function. Pharmacological inhibition of CaMKs as well as the genetic ablation of Camk4 reduced CREB phosphorylation and downregulated the expression of c-Fos, which is required for the induction of NFATc1 (the master transcription factor for osteoclastogenesis2,3) that is activated by receptor activator of NF-κB ligand (RANKL). Furthermore, CREB together with NFATc1 induced the expression of specific genes expressed by differentiated osteoclasts. Thus, the CaMK-CREB pathway biphasically functions to regulate the transcriptional program of osteoclastic bone resorption, by not only enhancing induction of NFATc1 but also facilitating NFATc1-dependent gene regulation once its expression is induced. This provides a molecular basis for a new therapeutic strategy for bone diseases.

Summary Ca 2+ signaling plays a central role in activity-dependent regulation of dendritic arborization, but key molecular mechanisms downstream of calcium elevation remain poorly understood. Here we show that the C-terminal region of the Ca 2+ /calmodulin-dependent protein kinase CLICK-III (CL3)/CaMKIγ, a membrane-anchored CaMK, was uniquely modified by two sequential lipidification steps: prenylation followed by a kinase-activity-regulated palmitoylation. These modifications were essential for CL3 membrane anchoring and targeting into detergent-resistant lipid microdomains (or rafts) in the dendrites. We found that CL3 critically contributed to BDNF-stimulated dendritic growth. Raft insertion of CL3 specifically promoted dendritogenesis of cortical neurons by acting upstream of RacGEF STEF and Rac, both present in lipid rafts. Thus, CL3 may represent a key element in the Ca 2+ -dependent and lipid-raft-delineated switch that turns on extrinsic activity-regulated dendrite formation in developing cortical neurons.

Postsynaptic density-95 (PSD-95), a PSD-95/Discs large/zona occludens-1 (PDZ) domain-containing scaffold protein, clusters many signaling molecules near NMDA-type glutamate receptors in the postsynaptic densities. Although the synaptic localization of PSD-95 requires palmitoylation of two cysteines at the N terminus and the presence of at least one PDZ domain, how the clustering of PSD-95 is initiated and regulated remains essentially unknown. To address this issue, we examined PSD-95 clustering in primary cultured hippocampal neurons expressing full-length PSD-95 mutant proteins lacking the ligand-binding ability of PDZ1, PDZ2, and/or PDZ3. The formation of either excitatory or inhibitory synapses was unaffected. Combinations of individual mutations, however, significantly reduced the PSD-95 clustering index, in an approximately additive manner. The sensitivity to 2-bromo-palmitate and latrunculin A, reagents known to affect PSD-95 turnover, was also augmented. Furthermore, the synaptic recruitment of a PSD-95 ligand, synaptic GTPase-activating protein (synGAP), was significantly impaired, whereas the clustering of other scaffolding proteins, such as Homer 1c, Shank/Synamon, and PSD-93/Chapsin-110 was spared. Intriguingly, overexpression of the PSD-95 PDZ1/2/3 mutants caused the PSD-95 clusters to localize away from the dendritic shaft, resulting in the formation of elongated spines, in an inverse correlation with the overall PDZ-ligand affinity. Expression of a mutant synGAP lacking the PDZ-binding motif replicated both the clustering and spine morphology phenotypes. In conclusion, the ligand-binding affinity of the PDZ domains of PSD-95, contributed in part via its interaction with the C-terminal end of synGAP, plays a critical role in titrating the synaptic clustering of PSD-95 and controlling its tight association with the PSD scaffold, thereby affecting synapse maturation.

Cytoskeleton is believed to contribute to activity-dependent processes underlying neuronal plasticity, such as regulations of cellular morphology and localization of signaling proteins. However, how neuronal activity controls actin cytoskeleton remains obscure. Taking advantage of confocal imaging of enhanced GFP-actin in the primary culture of hippocampal neurons, we show that synaptic activity induces multiple types of actin reorganization, both at the spines and at the somatic periphery. Activation of N -methyl- d -aspartate receptors, accompanied with a local rise in [Ca 2+ ]i, was sufficient to trigger a slow and sustained recruitment of actin into dendritic spines. In contrast, opening of voltage-gated Ca 2+ channels rapidly and reversibly enhanced cortical actin at the somatic periphery but not in the spines, in keeping with a high transient rise in somatic [Ca 2+ ]i. These data suggest that spatiotemporal dynamics of [Ca 2+ ]i, triggered by activation of N -methyl- d -aspartate receptors and voltage-gated Ca 2+ channels, provides the molecular basis for activity-dependent actin remodeling.

Corrigendum: Functional labeling of neurons and their projections using the synthetic activity–dependent promoter E-SARE