Inverse synaptic tagging of inactive synapses via dynamic interaction of Arc/Arg3.1 with CaMKIIß.

The 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 CaMKIIß 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 CaMKIIß 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.

Differential second messenger signaling via dopamine neurons bidirectionally regulates memory retention.

Memory formation and forgetting unnecessary memory must be balanced for adaptive animal behavior. While cyclic AMP (cAMP) signaling via dopamine neurons induces memory formation, here we report that cyclic guanine monophosphate (cGMP) signaling via dopamine neurons launches forgetting of unconsolidated memory in Drosophila. Genetic screening and proteomic analyses showed that neural activation induces the complex formation of a histone H3K9 demethylase, Kdm4B, and a GMP synthetase, Bur, which is necessary and sufficient for forgetting unconsolidated memory. Kdm4B/Bur is activated by phosphorylation through NO-dependent cGMP signaling via dopamine neurons, inducing gene expression, including kek2 encoding a presynaptic protein. Accordingly, Kdm4B/Bur activation induced presynaptic changes. Our data demonstrate a link between cGMP signaling and synapses via gene expression in forgetting, suggesting that the opposing functions of memory are orchestrated by distinct signaling via dopamine neurons, which affects synaptic integrity and thus balances animal behavior.

Arc controls alcohol cue relapse by a central amygdala mechanism.

Alcohol use disorder (AUD) is a chronic and fatal disease. The main impediment of the AUD therapy is a high probability of relapse to alcohol abuse even after prolonged abstinence. The molecular mechanisms of cue-induced relapse are not well established, despite the fact that they may offer new targets for the treatment of AUD. Using a comprehensive animal model of AUD, virally-mediated and amygdala-targeted genetic manipulations by CRISPR/Cas9 technology and ex vivo electrophysiology, we identify a mechanism that selectively controls cue-induced alcohol relapse and AUD symptom severity. This mechanism is based on activity-regulated cytoskeleton-associated protein (Arc)/ARG3.1-dependent plasticity of the amygdala synapses. In humans, we identified single nucleotide polymorphisms in the ARC gene and their methylation predicting not only amygdala size, but also frequency of alcohol use, even at the onset of regular consumption. Targeting Arc during alcohol cue exposure may thus be a selective new mechanism for relapse prevention.

Gastrin-releasing peptide regulates fear learning under stressed conditions via activation of the amygdalostriatal transition area.

The amygdala, a critical brain region responsible for emotional behavior, is crucially involved in the regulation of the effects of stress on emotional behavior. In the mammalian forebrain, gastrin-releasing peptide (GRP), a 27-amino-acid mammalian neuropeptide, which is a homolog of the 14-amino-acid amidated amphibian peptide bombesin, is highly expressed in the amygdala. The levels of GRP are markedly increased in the amygdala after acute stress; therefore, it is known as a stress-activated modulator. To determine the role of GRP in emotional behavior under stress, we conducted some behavioral and biochemical experiments with GRP-knockout (KO) mice. GRP-KO mice exhibited a longer freezing response than wild-type (WT) littermates in both contextual and auditory fear (also known as threat) conditioning tests only when they were subjected to acute restraint stress 20 min before the conditioning. To identify the critical neural circuits associated with the regulation of emotional memory by GRP, we conducted Arc/Arg3.1-reporter mapping in the amygdala with an Arc-Venus reporter transgenic mouse line. In the amygdalostriatal transition area (AST) and the lateral side of the basal nuclei, fear conditioning after restraint stress increased neuronal activity significantly in WT mice, and GRP KO was found to negate this potentiation only in the AST. These results indicate that the GRP-activated neurons in the AST are likely to suppress excessive fear expression through the regulation of downstream circuits related to fear learning following acute stress.

Direct Injection of Recombinant AAV-Containing Solution into the Oviductal Lumen of Pregnant Mice Caused In Situ Infection of Both Preimplantation Embryos and Oviductal Epithelium.

Adeno-associated virus (AAV) vector is an efficient viral-based gene delivery tool used with many types of cells and tissues, including neuronal cells and muscles. AAV serotype 6 (AAV-6), one of numerous AAV serotypes, was recently found to efficiently transduce mouse preimplantation embryos. Furthermore, through coupling with a clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system-a modern genome editing technology-AAV-6 has been shown to effectively create a mutation at a target locus, which relies on isolation of zygotes, in vitro viral infection, and transplantation of the infected embryos to recipient females. Unfortunately, this procedure, termed "ex vivo handling of embryos", requires considerable investment of capital, time, and effort. Direct transduction of preimplantation embryos through the introduction of AAV-6 into the oviductal lumen of pregnant females would be an ideal approach. In this study, we injected various types of recombinant AAV vectors (namely, rAAV-CAG-EGFP-1, -2, -5, and -6, each carrying an enhanced green fluorescent protein [EGFP] cDNA whose expression is under the influence of a cytomegalovirus enhancer + chicken ß-actin promoter) into the ampulla region of oviducts in pregnant female mice at Day 0.7 of pregnancy (corresponding to the late 1-cell stage), and EGFP-derived green fluorescence was assessed in the respective morulae. The highest levels of fluorescence were observed in rAAV-CAG-EGFP-6. The oviductal epithelium was distinctly fluorescent. The fluorescence in embryos peaked at the morula stage. Our results indicate that intra-oviductal injection of AAV-6 vectors is the most effective method for transducing zona pellucida-enclosed preimplantation embryos in situ. AAV-6 vectors could be a useful tool in the genetic manipulation of early embryos, as well as oviductal epithelial cells.

Quantification of native mRNA dynamics in living neurons using fluorescence correlation spectroscopy and reduction-triggered fluorescent probes.

RNA localization in subcellular compartments is essential for spatial and temporal regulation of protein expression in neurons. Several techniques have been developed to visualize mRNAs inside cells, but the study of the behavior of endogenous and nonengineered mRNAs in living neurons has just started. In this study, we combined reduction-triggered fluorescent (RETF) probes and fluorescence correlation spectroscopy (FCS) to investigate the diffusion properties of activity-regulated cytoskeleton-associated protein (Arc) and inositol 1,4,5-trisphosphate receptor type 1 (Ip3r1) mRNAs. This approach enabled us to discriminate between RNA-bound and unbound fluorescent probes and to quantify mRNA diffusion parameters and concentrations in living rat primary hippocampal neurons. Specifically, we detected the induction of Arc mRNA production after neuronal activation in real time. Results from computer simulations with mRNA diffusion coefficients obtained in these analyses supported the idea that free diffusion is incapable of transporting mRNA of sizes close to those of Arc or Ip3r1 to distal dendrites. In conclusion, the combined RETF-FCS approach reported here enables analyses of the dynamics of endogenous, unmodified mRNAs in living neurons, affording a glimpse into the intracellular dynamics of RNA in live cells.

Inverse synaptic tagging: An inactive synapse-specific mechanism to capture activity-induced Arc/arg3.1 and to locally regulate spatial distribution of synaptic weights.

Long-lasting forms of synaptic plasticity such as long-term potentiation (LTP) and long-term depression (LTD) are fundamental cellular mechanisms underlying learning and memory. The synaptic tagging and capture (STC) hypothesis has provided a theoretical framework on how products of activity-dependent genes may interact with potentiated synapses to facilitate and maintain such long-lasting synaptic plasticity. Although Arc/arg3.1 was initially assumed to participate in STC processes during LTP, accumulating evidence indicated that Arc/arg3.1 might rather contribute in weakening of synaptic weights than in their strengthening. In particular, analyses of Arc/Arg3.1 protein dynamics and function in the dendrites after plasticity-inducing stimuli have revealed a new type of inactivity-dependent redistribution of synaptic weights, termed "inverse synaptic tagging". The original synaptic tagging and inverse synaptic tagging likely co-exist and are mutually non-exclusive mechanisms, which together may help orchestrate the redistribution of synaptic weights and promote the enhancement and maintenance of their contrast between potentiated and non-potentiated synapses during the late phase of long-term synaptic plasticity. In this review, we describe the inverse synaptic tagging mechanism that controls synaptic dynamics of Arc/Arg3.1, an immediate early gene product which is captured and preferentially targeted to non-potentiated synapses, and discuss its impact on neuronal circuit refinement and cognitive function.

Arc restores juvenile plasticity in adult mouse visual cortex.

The molecular basis for the decline in experience-dependent neural plasticity over age remains poorly understood. In visual cortex, the robust plasticity induced in juvenile mice by brief monocular deprivation during the critical period is abrogated by genetic deletion of Arc, an activity-dependent regulator of excitatory synaptic modification. Here, we report that augmenting Arc expression in adult mice prolongs juvenile-like plasticity in visual cortex, as assessed by recordings of ocular dominance (OD) plasticity in vivo. A distinguishing characteristic of juvenile OD plasticity is the weakening of deprived-eye responses, believed to be accounted for by the mechanisms of homosynaptic long-term depression (LTD). Accordingly, we also found increased LTD in visual cortex of adult mice with augmented Arc expression and impaired LTD in visual cortex of juvenile mice that lack Arc or have been treated in vivo with a protein synthesis inhibitor. Further, we found that although activity-dependent expression of Arc mRNA does not change with age, expression of Arc protein is maximal during the critical period and declines in adulthood. Finally, we show that acute augmentation of Arc expression in wild-type adult mouse visual cortex is sufficient to restore juvenile-like plasticity. Together, our findings suggest a unifying molecular explanation for the age- and activity-dependent modulation of synaptic sensitivity to deprivation.

Deficiency of AMPAR-Palmitoylation Aggravates Seizure Susceptibility.

Synaptic AMPAR expression controls the strength of excitatory synaptic transmission and plasticity. An excess of synaptic AMPARs leads to epilepsy in response to seizure-inducible stimulation. The appropriate regulation of AMPARs plays a crucial role in the maintenance of the excitatory/inhibitory synaptic balance; however, the detailed mechanisms underlying epilepsy remain unclear. Our previous studies have revealed that a key modification of AMPAR trafficking to and from postsynaptic membranes is the reversible, posttranslational S-palmitoylation at the C-termini of receptors. To clarify the role of palmitoylation-dependent regulation of AMPARs in vivo, we generated GluA1 palmitoylation-deficient (Cys811 to Ser substitution) knock-in mice. These mutant male mice showed elevated seizure susceptibility and seizure-induced neuronal activity without impairments in synaptic transmission, gross brain structure, or behavior at the basal level. Disruption of the palmitoylation site was accompanied by upregulated GluA1 phosphorylation at Ser831, but not at Ser845, in the hippocampus and increased GluA1 protein expression in the cortex. Furthermore, GluA1 palmitoylation suppressed excessive spine enlargement above a certain size after LTP. Our findings indicate that an abnormality in GluA1 palmitoylation can lead to hyperexcitability in the cerebrum, which negatively affects the maintenance of network stability, resulting in epileptic seizures.SIGNIFICANCE STATEMENT AMPARs predominantly mediate excitatory synaptic transmission. AMPARs are regulated in a posttranslational, palmitoylation-dependent manner in excitatory synapses of the mammalian brain. Reversible palmitoylation dynamically controls synaptic expression and intracellular trafficking of the receptors. Here, we generated GluA1 palmitoylation-deficient knock-in mice to clarify the role of AMPAR palmitoylation in vivo We showed that an abnormality in GluA1 palmitoylation led to hyperexcitability, resulting in epileptic seizure. This is the first identification of a specific palmitoylated protein critical for the seizure-suppressing process. Our data also provide insight into how predicted receptors such as AMPARs can effectively preserve network stability in the brain. Furthermore, these findings help to define novel key targets for developing anti-epileptic drugs.

Involvement of SRF coactivator MKL2 in BDNF-mediated activation of the synaptic activity-responsive element in the Arc gene.

The expression of immediate early genes (IEGs) is thought to be an essential molecular basis of neuronal plasticity for higher brain function. Many IEGs contain serum response element in their transcriptional regulatory regions and their expression is controlled by serum response factor (SRF). SRF is known to play a role in concert with transcriptional cofactors. However, little is known about how SRF cofactors regulate IEG expression during the process of neuronal plasticity. We hypothesized that one of the SRF-regulated neuronal IEGs, activity-regulated cytoskeleton-associated protein (Arc; also termed Arg3.1), is regulated by an SRF coactivator, megakaryoblastic leukemia (MKL). To test this hypothesis, we initially investigated which binding site of the transcription factor or SRF cofactor contributes to brain-derived neurotrophic factor (BDNF)-induced Arc gene transcription in cultured cortical neurons using transfection and reporter assays. We found that BDNF caused robust induction of Arc gene transcription through a cAMP response element, binding site of myocyte enhancer factor 2, and binding site of SRF in an Arc enhancer, the synaptic activity-responsive element (SARE). Regardless of the requirement for the SRF-binding site, the binding site of a ternary complex factor, another SRF cofactor, did not affect BDNF-mediated Arc gene transcription. In contrast, chromatin immunoprecipitation revealed occupation of MKL at the SARE. Furthermore, knockdown of MKL2, but not MKL1, significantly decreased BDNF-mediated activation of the SARE. Taken together, these findings suggest a novel mechanism by which MKL2 controls the Arc SARE in response to BDNF stimulation.

Retained Plasticity and Substantial Recovery of Rod-Mediated Visual Acuity at the Visual Cortex in Blind Adult Mice with Retinal Dystrophy.

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 (Pde6ccpfl1/cpfl1Gnat1IRD2/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.

Neuromodulatory effect of Gαs- or Gαq-coupled G-protein-coupled receptor on NMDA receptor selectively activates the NMDA receptor/Ca2+/calcineurin/cAMP response element-binding protein-regulated transcriptional coactivator 1 pathway to effectively induce brain-derived neurotrophic factor expression in neurons.

Although coordinated molecular signaling through excitatory and modulatory neurotransmissions is critical for the induction of immediate early genes (IEGs), which lead to effective changes in synaptic plasticity, the intracellular mechanisms responsible remain obscure. Here we measured the expression of IEGs and used bioluminescence imaging to visualize the expression of Bdnf when GPCRs, major neuromodulator receptors, were stimulated. Stimulation of pituitary adenylate cyclase-activating polypeptide (PACAP)-specific receptor (PAC1), a Gαs/q-protein-coupled GPCR, with PACAP selectively activated the calcineurin (CN) pathway that is controlled by calcium signals evoked via NMDAR. This signaling pathway then induced the expression of Bdnf and CN-dependent IEGs through the nuclear translocation of CREB-regulated transcriptional coactivator 1 (CRTC1). Intracerebroventricular injection of PACAP and intraperitoneal administration of MK801 in mice demonstrated that functional interactions between PAC1 and NMDAR induced the expression of Bdnf in the brain. Coactivation of NMDAR and PAC1 synergistically induced the expression of Bdnf attributable to selective activation of the CN pathway. This CN pathway-controlled expression of Bdnf was also induced by stimulating other Gαs- or Gαq-coupled GPCRs, such as dopamine D1, adrenaline ß, CRF, and neurotensin receptors, either with their cognate agonists or by direct stimulation of the protein kinase A (PKA)/PKC pathway with chemical activators. Thus, the GPCR-induced expression of IEGs in coordination with NMDAR might occur via the selective activation of the CN/CRTC1/CREB pathway under simultaneous excitatory and modulatory synaptic transmissions in neurons if either the Gαs/adenylate cyclase/PKA or Gαq/PLC/PKC-mediated pathway is activated.

Class I histone deacetylase-mediated repression of the proximal promoter of the activity-regulated cytoskeleton-associated protein gene regulates its response to brain-derived neurotrophic factor.

We examined the transcriptional regulation of the activity-regulated cytoskeleton-associated protein gene (Arc), focusing on BDNF-induced Arc expression in cultured rat cortical cells. Although the synaptic activity-responsive element (SARE), located -7 kbp upstream of the Arc transcription start site, responded to NMDA, BDNF, or FGF2, the proximal region of the promoter (Arc/-1679) was activated by BDNF or FGF2, but not by NMDA, suggesting the presence of at least two distinct Arc promoter regions, distal and proximal, that respond to extracellular stimuli. Specificity protein 4 (SP4) and early growth response 1 (EGR1) controlled Arc/-1679 transcriptional activity via the region encompassing -169 to -37 of the Arc promoter. We found that trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, significantly enhanced the inductive effects of BDNF or FGF2, but not those of NMDA on Arc expression. Inhibitors of class I/IIb HDACs, SAHA, and class I HDACs, MS-275, but not of class II HDACs, MC1568, enhanced BDNF-induced Arc expression. The enhancing effect of TSA was mediated by the region from -1027 to -1000 bp, to which serum response factor (SRF) and HDAC1 bound. The binding of HDAC1 to this region was reduced by TSA. Thus, Arc expression was suppressed by class I HDAC-mediated mechanisms via chromatin modification of the proximal promoter whereas the inhibition of HDAC allowed Arc expression to be markedly enhanced in response to BDNF or FGF2. These results contribute to our understanding of the physiological role of Arc expression in neuronal functions such as memory consolidation.

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.

Tactile localization training for pain, sensory disturbance, and distorted body image: a case study of complex regional pain syndrome.

This report presents a case of complex regional pain syndrome. The patient presented with severe pain, sensory disturbance, and distorted body image at the site of initial injury and other body sites. Tactile localization training (TLT) at only the site of initial injury decreased severe pain at the site of initial injury and the secondary affected sites, whereas TLT at secondary affected sites had no effect. These results highlighted the importance of assessing changes in patients' pain processes to determine the part of the body where TLT should be applied.

Relative contribution of sensory and motor deficits on grip force control in patients with chronic stroke.

OBJECTIVE: This study aimed to characterize grasping behavior in static (weight-dependent modulation and stability of control) and dynamic (predictive control) aspects specifically focusing on the relative contribution of sensory and motor deficits to grip force control in patients with chronic stroke. METHODS: Twenty-four chronic stroke patients performed three manipulative tasks: five trials of 5-s grasp-lift-holding, 30-s static holding, and vertical dynamic/cyclic oscillation of holding the object. RESULTS: Exerted static grip force on the paretic side exhibited statistically greater than that on the non-paretic side. Spearman's rank correlation coefficient revealed that the contribution to static grip force control was larger in sensory deficits than in motor deficits. In addition, the sensory deficit is related to the reduced coupling between grip force and load force, suggesting difficulty in predictive control due to the absence of sensory feedback. CONCLUSIONS: Given that grip force control involves predictive feedforward and online feedback control, the evaluation of grip force might be an important and feasible evaluation manner for the assessment of sensorimotor control in patients post-stroke. SIGNIFICANCE: Detailed evaluation of grip force control would help to understand the mechanisms underlying hand dysfunction in stroke patients.

Orexin neurons play contrasting roles in itch and pain neural processing via projecting to the periaqueductal gray.

Pain and itch are recognized as antagonistically regulated sensations; pain suppresses itch, whilst pain inhibition enhances itch. The neural mechanisms at the central nervous system (CNS) underlying these pain-itch interactions still need to be explored. Here, we revealed the contrasting role of orexin-producing neurons (ORX neurons) in the lateral hypothalamus (LH), which suppresses pain while enhancing itch neural processing, by applying optogenetics to the acute pruritus and pain model. We also revealed that the circuit of ORX neurons from LH to periaqueductal gray regions served in the contrasting modulation of itch and pain processing using optogenetic terminal inhibition techniques. Additionally, by using an atopic dermatitis model, we confirmed the involvement of ORX neurons in regulating chronic itch processing, which could lead to a novel therapeutic target for persistent pruritus in clinical settings. Our findings provide new insight into the mechanism of antagonistic regulation between pain and itch in the CNS.

Changes in Standing Postural Control Ability in a Case of Spinocerebellar Ataxia Type 31 With Physical Therapy Focusing on the Center of Gravity Sway Variables and Lower Leg Muscle Activity.

Spinocerebellar degeneration (SCD) is a progressive disease characterized by cerebellar ataxia or the posterior spinal cord. Among these, spinocerebellar ataxia type 31 (SCA31) is genetically more common in the Japanese population and is characterized by pure ataxia, resulting in severe disturbances in postural balance, with common falls. Therefore, rehabilitation is important to improve postural balance. Light touch is a known method of reducing postural sway, which acts with the light touching of an object with the body. We herein present a case of a patient with SCA31 who was trained in a standing position by lightly touching the back of the body to a wall surface. Dynamic interarticular coordination exercises were also performed as part of the rehabilitation program. As a result, even in the progressive SCA31, improvements in standing postural balance and activities of daily living contributed to improvements in the patient's postural balance. We followed the progress of postural control ability using the center of gravity sway measurement and electromyography and described some interesting characteristics of the patient's postural control ability in this report.

Synaptic activity-responsive element in the Arc/Arg3.1 promoter essential for synapse-to-nucleus signaling in activated neurons.

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 approximately 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.