Satoshi Kamijo

Rational design of a high-affinity, fast, red calcium indicator R-CaMP2

Fluorescent Ca2+ 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 Ca2+ 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.

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

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.

Control of Cortical Axon Elongation by a GABA-Driven Ca2+/Calmodulin-Dependent Protein Kinase Cascade

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 GABAA receptor 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 vivo knockdown 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.

Region-specific activation of CRTC1-CREB signaling mediates long-term fear memory.

CREB is a pivotal mediator of activity-regulated gene transcription that underlies memory formation and allocation. The contribution of a key CREB cofactor, CREB-regulated transcription coactivator 1 (CRTC1), has, however, remained elusive. Here we show that several constitutive kinase pathways and an activity-regulated phosphatase, calcineurin, converge to determine the nucleocytoplasmic shuttling of CRTC1. This, in turn, triggered an activity-dependent association of CRTC1 with CREB-dependent regulatory elements found on IEG promoters. Forced expression of nuclear CRTC1 in hippocampal neurons activated CREB-dependent transcription, and was sufficient to enhance contextual fear memory. Surprisingly, during contextual fear conditioning, we found evidence of nuclear recruitment of endogenous CRTC1 only in the basolateral amygdala, and not in the hippocampus. Consistently, CRTC1 knockdown in the amygdala, but not in the hippocampus, significantly attenuated fear memory. Thus, CRTC1 has a wide impact on CREB-dependent memory processes, but fine-tunes CREB output in a region-specific manner.

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.

Differential roles for CaM kinases in mediating excitation–morphogenesis coupling during formation and maturation of neuronal circuits

Ca2+‐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 Ca2+ 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 critical neurodevelopmental role for L-type voltage-gated calcium channels in neurite extension and radial migration

Despite many association studies linking gene polymorphisms and mutations of L-type voltage-gated Ca2+ 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 Cav1.2 (CACNA1C) pore-forming subunit of L-type VGCCs, which were indeed expressed in immature mouse brains. Consistently, knocking out Cav1.2 resulted in significant alterations of neurite outgrowth. Furthermore, expression of a gain-of-function Cav1.2 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 Cav1.2 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 Ca2+ 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 Ca2+ indicator technology, quantitative pharmacology, and in utero electroporation and found a hitherto unsuspected role for L-type VGCCs in determining the Ca2+ 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.

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

In the version of this article initially published, in the Online Methods “RNase H libraries” section, the sentence beginning with “We added 5 μl preheated RNase H....” should have read “We added 5 μl preheated RNase H reaction mix that contains 10 U of Hybridase Thermostable RNase H (Epicentre), 0.5 μmol Tris-HCl, pH 7.5, 1 μmol NaCl and 0.2 μmol MgCl2 to the RNA and DNA oligo mix, incubated this mixture at 45 °C for 30 min and then placed it on ice.” The errors have been corrected in the HTML and PDF versions of this article.

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

In the version of this article initially published, in the Online Methods “RNase H libraries” section, the sentence beginning with “We added 5 μl preheated RNase H....” should have read “We added 5 μl preheated RNase H reaction mix that contains 10 U of Hybridase Thermostable RNase H (Epicentre), 0.5 μmol Tris-HCl, pH 7.5, 1 μmol NaCl and 0.2 μmol MgCl2 to the RNA and DNA oligo mix, incubated this mixture at 45 °C for 30 min and then placed it on ice.” The errors have been corrected in the HTML and PDF versions of this article.

Histological and behavioral analyses in CL3/CaMKIgamma-deficient mice

Neuronal activity in the dorsal raphé nucleus (DRN), a major source of serotonin, is modulated by the received reward size. To investigate whether DRN neurons code rewarding or aversive stimuli and/or positive or negative prediction error, we recorded single-unit activity in the DRN of two monkeys performing the trace conditioning task. This task consisted of two blocks with distinct contexts. In the appetitive block, liquid reward was used as an unconditioned stimulus (US). In the aversive block, air-puff directed at the monkey face was used as a negative US. In both blocks, three visual stimuli (conditioned stimuli: CSs) were paired with the US, with probabilities of 100, 50 and 0%, respectively. To confirm that monkeys learned the association of each specific CS with the US, we monitored licking behavior and anticipatory eye blinking. In 50 and 0% trials, tone was presented as a neutral stimulus in the absence of reward or air-puff. We recorded 211 task-related neurons. It was found that DRN neurons responded to the CSs in the appetitive block more often than in the aversive block; 38% (n = 81) responded to the rewarding CS with 100% probability, while 9% (n = 19) responded to the aversive CS with 100% probability. Among them, 13 neurons responded to both rewarding and aversive CSs. Many DRN neurons also responded to the USs (n = 176); either to reward only (n = 35), to air-puff only (n = 57) or to both (n = 84). In the appetitive block, rewardrelated activity was modulated by its probability with stronger response to the unpredicted than the predicted US. In the aversive block, short-latency response to air-puff delivery was frequently observed regardless of the CS-US predictability. These results suggest that the primate DRN codes information about both rewarding and aversive stimuli, and some DRN neurons exhibited activity similar to reward prediction error reported for dopamine neurons.