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.

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.

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.

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.

Prolonged contextual fear memory in AMPA receptor palmitoylation-deficient mice.

Long-lasting fear-related disorders depend on the excessive retention of traumatic fear memory. We previously showed that the palmitoylation-dependent removal of synaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors prevents hyperexcitation-based epileptic seizures and that AMPA receptor palmitoylation maintains neural network stability. In this study, AMPA receptor subunit GluA1 C-terminal palmitoylation-deficient (GluA1C811S) mice were subjected to comprehensive behavioral battery tests to further examine whether the mutation causes other neuropsychiatric disease-like symptoms. The behavioral analyses revealed that palmitoylation-deficiency in GluA1 is responsible for characteristic prolonged contextual fear memory formation, whereas GluA1C811S mice showed no impairment of anxiety-like behaviors at the basal state. In addition, fear generalization gradually increased in these mutant mice without affecting their cued fear. Furthermore, fear extinction training by repeated exposure of mice to conditioned stimuli had little effect on GluA1C811S mice, which is in line with augmentation of synaptic transmission in pyramidal neurons in the basolateral amygdala. In contrast, locomotion, sociability, depression-related behaviors, and spatial learning and memory were unaffected by the GluA1 non-palmitoylation mutation. These results indicate that impairment of AMPA receptor palmitoylation specifically causes posttraumatic stress disorder (PTSD)-like symptoms.

Claustrum mediates bidirectional and reversible control of stress-induced anxiety responses.

The processing of stress responses involves brain-wide communication among cortical and subcortical regions; however, the underlying mechanisms remain elusive. Here, we show that the claustrum (CLA) is crucial for the control of stress-induced anxiety-related behaviors. A combined approach using brain activation mapping and machine learning showed that the CLA activation serves as a reliable marker of exposure to acute stressors. In TRAP2 mice, which allow activity-dependent genetic labeling, chemogenetic activation of the CLA neuronal ensemble tagged by acute social defeat stress (DS) elicited anxiety-related behaviors, whereas silencing of the CLA ensemble attenuated DS-induced anxiety-related behaviors. Moreover, the CLA received strong input from DS-activated basolateral amygdala neurons, and its circuit-selective optogenetic photostimulation temporarily elicited anxiety-related behaviors. Last, silencing of the CLA ensemble during stress exposure increased resistance to chronic DS. The CLA thus bidirectionally controls stress-induced emotional responses, and its inactivation can serve as a preventative strategy to increase stress resilience.

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.

Functional correlates of immediate early gene expression in mouse visual cortex.

During visual development, response properties of layer 2/3 neurons in visual cortex are shaped by experience. Both visual and visuomotor experience are necessary to co-ordinate the integration of bottom-up visual input and top-down motor-related input. Whether visual and visuomotor experience engage different plasticity mechanisms, possibly associated with the two separate input pathways, is still unclear. To begin addressing this, we measured the expression level of three different immediate early genes (IEG) (c-fos, egr1 or Arc) and neuronal activity in layer 2/3 neurons of visual cortex before and after a mouse's first visual exposure in life, and subsequent visuomotor learning. We found that expression levels of all three IEGs correlated positively with neuronal activity, but that first visual and first visuomotor exposure resulted in differential changes in IEG expression patterns. In addition, IEG expression levels differed depending on whether neurons exhibited primarily visually driven or motor-related activity. Neurons with strong motor-related activity preferentially expressed EGR1, while neurons that developed strong visually driven activity preferentially expressed Arc. Our findings are consistent with the interpretation that bottom-up visual input and top-down motor-related input are associated with different IEG expression patterns and hence possibly also with different plasticity pathways.

Cooperation of LIM domain-binding 2 (LDB2) with EGR in the pathogenesis of schizophrenia.

Genomic defects with large effect size can help elucidate unknown pathologic architecture of mental disorders. We previously reported on a patient with schizophrenia and a balanced translocation between chromosomes 4 and 13 and found that the breakpoint within chromosome 4 is located near the LDB2 gene. We show here that Ldb2 knockout (KO) mice displayed multiple deficits relevant to mental disorders. In particular, Ldb2 KO mice exhibited deficits in the fear-conditioning paradigm. Analysis of the amygdala suggested that dysregulation of synaptic activities controlled by the immediate early gene Arc is involved in the phenotypes. We show that LDB2 forms protein complexes with known transcription factors. Consistently, ChIP-seq analyses indicated that LDB2 binds to > 10,000 genomic sites in human neurospheres. We found that many of those sites, including the promoter region of ARC, are occupied by EGR transcription factors. Our previous study showed an association of the EGR family genes with schizophrenia. Collectively, the findings suggest that dysregulation in the gene expression controlled by the LDB2-EGR axis underlies a pathogenesis of subset of mental disorders.

Rpd3/CoRest-mediated activity-dependent transcription regulates the flexibility in memory updating in Drosophila.

Consolidated memory can be preserved or updated depending on the environmental change. Although such conflicting regulation may happen during memory updating, the flexibility of memory updating may have already been determined in the initial memory consolidation process. Here, we explored the gating mechanism for activity-dependent transcription in memory consolidation, which is unexpectedly linked to the later memory updating in Drosophila. Through proteomic analysis, we discovered that the compositional change in the transcriptional repressor, which contains the histone deacetylase Rpd3 and CoRest, acts as the gating mechanism that opens and closes the time window for activity-dependent transcription. Opening the gate through the compositional change in Rpd3/CoRest is required for memory consolidation, but closing the gate through Rpd3/CoRest is significant to limit future memory updating. Our data indicate that the flexibility of memory updating is determined through the initial activity-dependent transcription, providing a mechanism involved in defining memory state.

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.

GABAergic neurons in the olfactory cortex projecting to the lateral hypothalamus in mice.

Olfaction guides goal-directed behaviours including feeding. To investigate how central olfactory neural circuits control feeding behaviour in mice, we performed retrograde tracing from the lateral hypothalamus (LH), an important feeding centre. We observed a cluster of retrogradely labelled cells distributed in the posteroventral region of the olfactory peduncle. Histochemical analyses revealed that the majority of these retrogradely labelled projection neurons expressed glutamic acid decarboxylase 65/67 (GAD65/67), but not vesicular glutamate transporter 1 (VGluT1). We named this region containing GABAergic projection neurons the ventral olfactory nucleus (VON) to differentiate it from the conventional olfactory peduncle. VON neurons were less immunoreactive for DARPP-32, a striatal neuron marker, compared to neurons in the olfactory tubercle and nucleus accumbens, which distinguished the VON from the ventral striatum. Fluorescent labelling confirmed putative synaptic contacts between VON neurons and olfactory bulb projection neurons. Rabies-virus-mediated trans-synaptic labelling revealed that VON neurons received synaptic inputs from the olfactory bulb, other olfactory cortices, horizontal limb of the diagonal band, and prefrontal cortex. Collectively, these results identify novel GABAergic projection neurons in the olfactory cortex that may integrate olfactory sensory and top-down inputs and send inhibitory output to the LH, which may modulate odour-guided LH-related behaviours.

A clickable caging group as a new platform for modular caged compounds with improved photochemical properties.

A 6-bromo-7-hydroxycoumarin-4-ylmethyl (Bhc) caged compound having a click-modifiable chemical handle was designed and synthesized. This molecule was applied to the synthesis of modular caged paclitaxels (PTXs) in which additional functional units could be easily installed. This system was used to prepare water-soluble caged PTXs with improved photolysis efficiencies.

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.

Perturbed expression pattern of the immediate early gene Arc in the dentate gyrus of GluA1 C-terminal palmitoylation-deficient mice.

BACKGROUND: AMPA receptors predominantly mediate fast excitatory synaptic transmission in the mammalian brain. Post-translational protein S-palmitoylation of AMPA receptor GluA subunits at their C-termini reversibly controls the receptors trafficking to and from excitatory glutamatergic synapses. Excitatory inputs to neurons induce the expression of immediate early genes (IEGs), including Arc, with particular spatial patterns. In the hippocampal dentate gyrus, Arc is mainly expressed in the upper (dorsal) blade at the basal state. GluA1 C-terminal palmitoylation-deficient (GluA1C811S) mice showed enhanced seizure susceptibility and disturbed synaptic plasticity without impaired gross anatomy or basal synaptic transmission. These mutant mice also exhibited an increased expression of IEG products, c-Fos and Arc proteins, in the hippocampus and cerebral cortex. In this report, we further analyzed excitability and Arc expression pattern in the dentate gyrus of GluA1C811S mice. METHODS AND RESULTS: Electrophysiological analysis of granule neurons to measure the evoked excitatory postsynaptic current/evoked inhibitory postsynaptic current ratio revealed that excitatory/inhibitory (E/I) balance was normal in GluA1C811S mice. In contrast, immunohistochemical staining showed an abnormal distribution of Arc-positive cells between upper and lower (ventral) blades of the dentate gyrus in these mutant mice. These data suggest that deficiency of GluA1 palmitoylation causes perturbed neuronal inputs from the entorhinal cortex to the dentate gyrus, which potentially underlies the excessive excitability in response to seizure-inducing stimulation. CONCLUSION: Our findings conclude that an appropriate regulation of Arc expression in the dentate gyrus, ensured by AMPA receptor palmitoylation, may be critical for stabilizing hippocampal neural circuits and may suppress excess excitation.

Seizure-Induced Arc mRNA Expression Thresholds in Rat Hippocampus and Perirhinal Cortex.

Immediate-early genes (IEGs) are rapidly and transiently induced following excitatory neuronal activity including maximal electroconvulsive shock treatment (ECT). The rapid RNA response can be blocked by the sodium channel antagonist tetrodotoxin (TTX), without blocking seizures, indicating a role for electrical stimulation in electroconvulsive shock-induced mRNA responses. In behaving animals, Arc mRNA is selectively transcribed following patterned neuronal activity and rapidly trafficked to dendrites where it preferentially accumulates at active synapses for local translation. Here we examined whether there is a relationship between the current intensities that elicit seizures and the threshold for Arc mRNA transcription in the rat hippocampus and perirhinal cortex (PRC). Animals received ECT of varying current intensities (0, 20, 40 65, 77 and 85 mA) and were sacrificed 5 min later. While significantly more CA1, CA3 and perirhinal pyramidal cells expressed Arc at the lowest stimulus intensity compared to granule cells, there was an abrupt threshold transition that occurred in all four regions at 77 mA. This precise threshold for Arc expression in all temporal lobe neurons examined may involve regulation of the calcium-dependent mechanisms that are upstream to activity-dependent IEG transcription.

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.