Gene expression and its network structure are dynamically altered in multicellular systems during morphological, functional, and pathological changes. To precisely analyze the functional roles of dynamic gene expression changes, tools that manipulate gene expression at fine spatiotemporal resolution are needed. The tetracycline (Tet)-controlled gene expression system is a reliable drug-inducible method, and it is used widely in many mammalian cultured cells and model organisms. Here, we develop a photoactivatable (PA)-Tet-OFF/ON system for precise temporal control of gene expression at single-cell resolution. By integrating the cryptochrome 2-cryptochrome-interacting basic helix-loop-helix 1 (Cry2-CIB1) light-inducible binding switch, expression of the gene of interest is tightly regulated under the control of light illumination and drug application in our PA-Tet-OFF/ON system. This system has a large dynamic range of downstream gene expression and rapid activation/deactivation kinetics. We also demonstrate the optogenetic regulation of exogenous gene expression in vivo, such as in developing and adult mouse brains.

Limb apraxia is a higher brain dysfunction that typically occurs after left hemispheric stroke and its cause cannot be explained by sensory disturbance or motor paralysis. The comparison of motor signals and visual feedback to generate errors, i.e., visuo-motor integration, is important in motor control and motor learning, which may be impaired in apraxia. However, in apraxia after stroke, it is unknown whether there is a specific deficit in visuo-motor temporal integration compared to visuo-tactile and visuo-proprioceptive temporal integration. We examined the precision of visuo-motor temporal integration and sensory-sensory (visuo-tactile and visuo-proprioception) temporal integration in apraxia after stroke by using a delayed visual feedback detection task with three different conditions (tactile, passive movement, and active movement). The delay detection threshold and the probability curve for delay detection obtained in this task were quantitative indicators of the respective temporal integration functions. In addition, we performed subtraction and voxel-based lesion-symptom mapping to identify the brain lesions responsible for apraxia and deficits in visuo-motor temporal integration. The behavioral experiments showed that the delay detection threshold was extended and that the probability curve for delay detection was less steep in apraxic patients compared to controls (pseudo-apraxic patients and unaffected patients), only for the active movement condition, and not for the tactile and passive movement conditions. Furthermore, the severity of apraxia was significantly correlated with the delay detection threshold and the steepness of the probability curve in the active movement condition. These results indicated that multisensory (i.e., visual, tactile, and proprioception) feedback was normally temporally integrated, but motor prediction and visual feedback were not correctly temporally integrated in apraxic patients. That is, apraxic patients had difficulties with visuo-motor temporal integration. Lesion analyses revealed that both apraxia and the distortion of visuo-motor temporal integration were associated with lesions in the fronto-parietal motor network, including the left inferior parietal lobule and left inferior frontal gyrus. We suppose that damage to the left inferior fronto-parietal network could cause deficits in motor prediction for visuo-motor temporal integration, but not for sensory-sensory (visuo-tactile and visuo-proprioception) temporal integration, leading to the distortion of visuo-motor temporal integration in patients with apraxia.

Odour information induces various innate responses that are critical to the survival of the individual and for the species. An axon guidance molecule, Neuropilin 2 (Nrp2), is known to mediate targeting of olfactory sensory neurons (primary neurons), to the posteroventral main olfactory bulb (PV MOB) in mice. Here we report that Nrp2-positive (Nrp2+) mitral cells (MCs, second-order neurons) play crucial roles in transmitting attractive social signals from the PV MOB to the anterior part of medial amygdala (MeA). Semaphorin 3F, a repulsive ligand to Nrp2, regulates both migration of Nrp2+ MCs to the PV MOB and their axonal projection to the anterior MeA. In the MC-specific Nrp2 knockout mice, circuit formation of Nrp2+ MCs and odour-induced attractive social responses are impaired. In utero, electroporation demonstrates that activation of the Nrp2 gene in MCs is sufficient to instruct their circuit formation from the PV MOB to the anterior MeA.

N-methyl-D-aspartate receptors (NMDARs) are critical for neuronal development and synaptic plasticity. Dysregulation of NMDARs is implicated in neuropsychiatric disorders. Native NMDARs are heteromultimeric protein complexes consisting of NR1 and NR2 subunits. NR2 subunits (NR2A-D) are the major determinants of the functional properties of NMDARs. Most research has focused on NR2A- and/or NR2B-containing receptors. A recent study demonstrated that NR2C- and/or NR2D-containing NMDARs are the primary targets of memantine, a drug that is widely prescribed to treat Alzheimer's disease. Our laboratory demonstrated that memantine prevents the loss of retinal ganglion cells (RGCs) in GLAST glutamate transporter knockout mice, a model of normal tension glaucoma (NTG), suggesting that NR2D-containing receptors may be involved in RGC loss in NTG.

Fear is one of the most potent emotional experiences and is an adaptive component of response to potentially threatening stimuli. Cumulative evidence suggests that the amygdala plays a central role in the acquisition, storage and expression of fear memory. We previously showed that the selective ablation of striatal neurons in the adult brain impairs the long-term, but not short-term, memory for auditory fear conditioning with a lower-intensity footshock. This finding raises an intriguing possibility that long-term auditory fear memory may be consolidated in the striatum.

Interleukin-1 receptor accessory protein-like 1 (IL1RAPL1) is associated with X-linked mental retardation and autism spectrum disorder. We found that IL1RAPL1 regulates synapse formation of cortical neurons. To investigate how IL1RAPL1 controls synapse formation, we here screened IL1RAPL1-interacting proteins by affinity chromatography and mass spectroscopy. IL1RAPL1 interacted with Mcf2-like (Mcf2l), a Rho guanine nucleotide exchange factor, through the cytoplasmic Toll/IL-1 receptor domain. Knockdown of endogenous Mcf2l and treatment with an inhibitor of Rho-associated protein kinase (ROCK), the downstream kinase of RhoA, suppressed IL1RAPL1-induced excitatory synapse formation of cortical neurons. Furthermore, we found that the expression of IL1RAPL1 affected the turnover of AMPA receptor subunits. Insertion of GluA1-containing AMPA receptors to the cell surface was decreased, whereas that of AMPA receptors composed of GluA2/3 was enhanced. Mcf2l knockdown and ROCK inhibitor treatment diminished the IL1RAPL1-induced changes of AMPA receptor subunit insertions. Our results suggest that Mcf2l-RhoA-ROCK signaling pathway mediates IL1RAPL1-dependent formation and stabilization of glutamatergic synapses of cortical neurons.

Neural circuits are shaped by activity-dependent elimination of redundant synapses during postnatal development. In many systems, postsynaptic activity is known to be crucial, but the precise mechanisms remain elusive. Here, we report that the immediate early gene Arc/Arg3.1 mediates elimination of surplus climbing fiber (CF) to Purkinje cell (PC) synapses in the developing cerebellum. CF synapse elimination was accelerated when activity of channelrhodopsin-2-expressing PCs was elevated by 2-day photostimulation. This acceleration was suppressed by PC-specific knockdown of either the P/Q-type voltage-dependent Ca(2+) channels (VDCCs) or Arc. PC-specific Arc knockdown had no appreciable effect until around postnatal day 11 but significantly impaired CF synapse elimination thereafter, leaving redundant CF terminals on PC somata. The effect of Arc knockdown was occluded by simultaneous knockdown of P/Q-type VDCCs in PCs. We conclude that Arc mediates the final stage of CF synapse elimination downstream of P/Q-type VDCCs by removing CF synapses from PC somata.

Phencyclidine (PCP), a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, increases locomotor activity in rodents and causes schizophrenia-like symptoms in humans. Although activation of the dopamine (DA) pathway is hypothesized to mediate these effects of PCP, the precise mechanisms by which PCP induces its effects remain to be elucidated. The present study investigated the effect of PCP on extracellular levels of DA (DA(ex)) in the striatum and prefrontal cortex (PFC) using in vivo microdialysis in mice lacking the NMDA receptor channel ε1 or ε4 subunit (GluRε1 [GluN2A] or GluRε4 [GluN2D]) and locomotor activity. PCP significantly increased DA(ex) in wildtype and GluRε1 knockout mice, but not in GluRε4 knockout mice, in the striatum and PFC. Acute and repeated administration of PCP did not increase locomotor activity in GluRε4 knockout mice. The present results suggest that PCP enhances dopaminergic transmission and increases locomotor activity by acting at GluRε4.

Fear is one of the most potent emotional experiences and is an adaptive component of response to potentially threatening stimuli. On the other hand, too much or inappropriate fear accounts for many common psychiatric problems. Cumulative evidence suggests that the amygdala plays a central role in the acquisition, storage and expression of fear memory. Here, we developed an inducible striatal neuron ablation system in transgenic mice. The ablation of striatal neurons in the adult brain hardly affected the auditory fear learning under the standard condition in agreement with previous studies. When conditioned with a low-intensity unconditioned stimulus, however, the formation of long-term fear memory but not short-tem memory was impaired in striatal neuron-ablated mice. Consistently, the ablation of striatal neurons 24 h after conditioning with the low-intensity unconditioned stimulus, when the long-term fear memory was formed, diminished the retention of the long-term memory. Our results reveal a novel form of the auditory fear memory depending on striatal neurons at the low-intensity unconditioned stimulus.

The semaphorin gene family contains a large number of secreted and transmembrane proteins; some function as repulsive and attractive cues of axon guidance during development. Here, we report cloning and characterization of zebrafish transmembrane semaphorin gene, semaphorin 6D (sema6D). Sema6D is expressed predominantly in the nervous system during embryogenesis, as determined by in situ hybridization. We also found that Sema6D binds Plexin-A1 in vitro, but not other Plexins. It induces the repulsion of dorsal root ganglion axons, but not sympathetic axons. Consequently, Sema6D might use Plexin-A1 as a receptor to repel specific types of axons during development.

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.

Neural network remodeling underpins the ability to remember life experiences, but little is known about the long-term plasticity of neural populations. To study how the brain encodes episodic events, we used time-lapse two-photon microscopy and a fluorescent reporter of neural plasticity based on an enhanced form of the synaptic activity-responsive element (E-SARE) within the Arc promoter to track thousands of CA1 hippocampal pyramidal cells over weeks in mice that repeatedly encountered different environments. Each environment evokes characteristic patterns of ensemble neural plasticity, but with each encounter, the set of activated cells gradually evolves. After repeated exposures, the plasticity patterns evoked by an individual environment progressively stabilize. Compared with young adults, plasticity patterns in aged mice are less specific to individual environments and less stable across repeat experiences. Long-term consolidation of hippocampal plasticity patterns may support long-term memory formation, whereas weaker consolidation in aged subjects might reflect declining memory function.

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.

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.

Synapse formation is triggered through trans-synaptic interaction between pairs of pre- and postsynaptic adhesion molecules, the specificity of which depends on splice inserts known as 'splice-insert signaling codes'. Receptor protein tyrosine phosphatase δ (PTPδ) can bidirectionally induce pre- and postsynaptic differentiation of neurons by trans-synaptically binding to interleukin-1 receptor accessory protein (IL-1RAcP) and IL-1RAcP-like-1 (IL1RAPL1) in a splicing-dependent manner. Here, we report crystal structures of PTPδ in complex with IL1RAPL1 and IL-1RAcP. The first immunoglobulin-like (Ig) domain of IL1RAPL1 directly recognizes the first splice insert, which is critical for binding to IL1RAPL1. The second splice insert functions as an adjustable linker that positions the Ig2 and Ig3 domains of PTPδ for simultaneously interacting with the Ig1 domain of IL1RAPL1 or IL-1RAcP. We further identified the IL1RAPL1-specific interaction, which appears coupled to the first-splice-insert-mediated interaction. Our results thus reveal the decoding mechanism of splice-insert signaling codes for synaptic differentiation induced by trans-synaptic adhesion between PTPδ and IL1RAPL1/IL-1RAcP.

The present study aimed to investigate the involvement of the NMDA receptor (NMDAR) and/or nitric oxide (NO) pathway in ketamine-induced behavioral sensitization. Mice received repeated subcutaneous administration of ketamine (25mg/kg), once daily or once weekly for a total of five doses. Even three administrations of ketamine, daily or weekly, induced a rapid increase in locomotor activity in wild-type (WT), but not in GluN2D knockout (GluN2D-KO) mice. Furthermore, for WT mice receiving daily ketamine, elevated locomotor activity was maintained after a 1-month withdrawal period; however, this was not the case when ketamine was administered weekly. The effect of acute ketamine on nNOS activities was estimated with nicotinamide adenine dinucleotide hydrogen phosphate-diaphorase (NADPH-d) histochemistry. Ketamine rapidly increased the number of NADPH-d activated cells and strongly stained dendrites in the dorsal striatum and prefrontal cortex of WT mice, but not GluN2D-KO mice. These results suggest that ketamine-induced locomotor sensitization and nNOS activation in the frontal cortex-striatum neuronal circuit are positively correlated and that the NMDAR GluN2D subunit plays an important role in the acquisition and maintenance of ketamine-induced behavioral sensitization.

Zebrafish is one of the best model organisms for investigating gene functions in vertebrates. By 4,5',8-trimethylpsoralen mutagenesis, we isolated a zebrafish mutant, vibrato, with defects in the spontaneous contraction and touch response. Whole genome subtraction between the wild-type and the mutant genomes by representational difference analysis yielded polymorphic markers tightly linked to the vibrato locus. Using these markers, we constructed a high-resolution physical map and localized the vibrato locus within a genomic region of 720 kb. Direct cDNA selection with the contig led to the identification of a novel gene, solo, encoding a protein with SEC14 and spectrin repeat domains. These domains of Solo shared significant amino acid sequence identities with those of mammalian Trio and Karilin. In addition, we found the zebrafish orthologs for mammalian TTN, COL5A2, and CED-6 in the vibrato region. Mapping of these genes localized human chromosomal regions possibly involved in motor disorders. Our results suggest that representational difference analysis provides an efficient way to isolate mutated genomic regions in zebrafish.

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.

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.

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.