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BACKGROUNDOne of the critical unmet medical needs in schizophrenia is a remedy for cognitive deficits. However, the neural circuit mechanisms of them remain unresolved. In addition, despite the patients with schizophrenia cannot stop taking antipsychotics due to a high rate of discontinuation-induced relapse, previous studies using animal models of schizophrenia have not considered these clinical situations.METHODSHere, we employ multi-dimensional approaches, including histological analysis in the prelimbic cortex, LC-MS/MS-based in vivo dopamine D2 receptor occupancy analysis for antipsychotic drugs, in vivo calcium imaging and behavioral analyses of mice using chemogenetic manipulation, to investigate neural mechanisms and potential therapeutic interventions for working memory deficit in a mouse model with chronic phencyclidine (PCP) administration that resembles the schizophrenia symptomatology.RESULTSChronic PCP administration led to abnormalities in excitatory and inhibitory synapses, including dendritic spines of pyramidal neurons, vesicular glutamate transporter 1 (VGLUT1) positive terminals, and parvalbumin (PV) positive GABAergic interneurons, in layer 2–3 of the prelimbic cortex. Continuous olanzapine, which achieved a sustained therapeutic window of dopamine D2 receptor occupancy (60–80%) in the striatum, did not affect these synaptic abnormalities and working memory deficit in the PCP-treated mice. We found that the selective prelimbic PV activation, using hM3D(Gq)-DREADD system confirmed by in vivo calcium imaging, restored working memory deficit, even under continuous olanzapine treatment.CONCLUSIONSOur study raises a possibility that intervention in prefrontal PV neurons leads to an add-on therapy to antipsychotics targeting amelioration of treatment-resistant cognitive deficits in schizophrenia.

The MicroLED probe enables optogenetic control of neural activity in spatially separated brain regions. Understanding its heat generation characteristics is important. In this study, we investigated the temperature rise (ΔT) characteristics in the brain tissue using a MicroLED probe. The ΔT strongly depended on the surrounding environment of the probe, including the differences between the air and the brain, and the area touching the brain tissue. Through animal experiments, we suggest an in situ temperature monitoring method using temperature dependence on electrical characteristics of the MicroLED. Finally, optical stimulation by MicroLEDs proved effective in controlling optogenetic neural activity in animal models.

Characterization of inter-regional interactions in brain is essential for understanding the mechanism relevant to normal brain function and neurological disease. The recently developed flexible micro (μ)-electrocorticography (μECoG) device is one prominent method used to examine large-scale cortical activity across multiple regions. The sheet-shaped μECoG electrodes arrays can be placed on a relatively wide area of cortical surface beneath the skull by inserting the device into the space between skull and brain. Although rats and mice are useful tools for neuroscience, current μECoG recording methods in these animals are limited to the parietal region of cerebral cortex. Recording cortical activity from the temporal region of cortex in mice has proven difficult because of surgical barriers created by the skull and surrounding temporalis muscle anatomy. Here, we developed a sheet-shaped 64-channel μECoG device that allows access to the mouse temporal cortex, and we determined the factor determining the appropriate bending stiffness for the μECoG electrode array. We also established a surgical technique to implant the electrode arrays into the epidural space over a wide area of cerebral cortex covering from the barrel field to olfactory (piriform) cortex, which is the deepest region of the cerebral cortex. Using histology and computed tomography (CT) images, we confirmed that the tip of the μECoG device reached to the most ventral part of cerebral cortex without causing noticeable damage to the brain surface. Moreover, the device simultaneously recorded somatosensory and odor stimulus-evoked neural activity from dorsal and ventral parts of cerebral cortex in awake and anesthetized mice. These data indicate that our μECoG device and surgical techniques enable the recording of large-scale cortical activity from the parietal to temporal cortex in mice, including somatosensory and olfactory cortices. This system will provide more opportunities for the investigation of physiological functions from wider areas of the mouse cerebral cortex than those currently available with existing ECoG techniques.

Currently, calcium imaging allows for the long-term recording of large-scale neuronal activity in diverse states. However, it remains difficult to extract neuronal dynamics from recorded imaging data. In this study, we propose an improved CNMF-based algorithm and an effective method for extracting cell shapes with fewer false positives and false negatives caused by image processing. We also showed that the values obtained during image processing can be combined and used for false positive determination of cells. For the CNMF algorithm, we combined cell-by-cell regularization and baseline shrinkage estimation, which greatly improved its stability and robustness. We applied these methods to artificial and real data and confirmed their effectiveness. Our method is simpler and faster, detects more cells with lower firing rates and signal-to-noise ratios, and enhance the quality of the extracted cell signals. These advances can improve the standard of downstream analysis and contribute to progress in neuroscience.

SummaryWhen processing current sensory inputs, animals refer to related past experiences. Current information is then incorporated into the related neural network to update previously stored memories. However, the neuronal mechanism underlying the impact of memories of prior experiences on current learning is not well understood. Here, we found that a cellular ensemble in the posterior parietal cortex (PPC) that is activated during past experience mediates an interaction between past and current information to update memory through a PPC-anterior cingulate cortex circuit in mice. Moreover, optogenetic silencing of the PPC ensemble immediately after retrieval dissociated the interaction without affecting individual memories stored in the hippocampus and amygdala. Thus, a specific subpopulation of PPC cells represents past information and instructs downstream brain regions to update previous memories.

A development of a biocompatible, optical stimulation device capable of adhering to the brain surface and activating spatially separated brain regions is necessary for in vivo optogenetic applications. In this study, a hollow structure for isolating the microLED epitaxial layer was fabricated using the anisotropic KOH wet-etching method. Using a thermal release sheet, a method to transfer microLEDs onto a biocompatible parylene film was established without rotation or misalignment of the microLEDs while retaining their characteristics. Accordingly, a flexible microLED array film was fabricated, which adhered to the surface of the brain of a mouse and exhibited blue emission.

Previous gain-of-function studies using an optogenetic technique showed that manipulation of the hippocampal dentate gyrus or CA1 cell ensembles is important for memory reactivation and to generate synthetic or false memory. However, gain-of-function study manipulating CA3 cell ensembles has not been reported. The CA3 area of the hippocampus comprises a recurrent excitatory circuit, which is thought to be important for the generation of associations among the stored information within one brain region. We investigated whether the coincident firing of cell ensembles in one brain region, hippocampal CA3, associates distinct events. CA3 cell ensembles responding to context exploration and during contextual fear conditioning were labeled with channelrhodopsin-2 (ChR2)-mCherry. The synchronous activation of these ensembles induced freezing behavior in mice in a neutral context, in which a foot shock had never been delivered. The recall of this artificial associative fear memory was context specific. In vivo electrophysiological recordings showed that 20-Hz optical stimulation of ChR2-mCherry-expressing CA3 neurons, which is the same stimulation protocol used in behavioral experiment, induced long-term potentiation at CA3-CA3 synapses. Altogether, these results demonstrate that the synchronous activation of ensembles in one brain region, CA3 of the hippocampus, is sufficient for the association of distinct events. The results of our electrophysiology potentially suggest that this artificial association of memory events might be induced by the strengthening of synaptic efficacy between CA3 ensembles via recurrent circuit. Electronic supplementary material The online version of this article (10.1186/s13041-018-0424-1) contains supplementary material, which is available to authorized users.

The hippocampus is crucial for declarative memories in humans and encodes episodic and spatial memories in animals. Memory coding strengthens synaptic efficacy via an LTP-like mechanism. Given that animals store memories of everyday experiences, the hippocampal circuit must have a mechanism that prevents saturation of overall synaptic weight for the preservation of learning capacity. LTD works to balance plasticity and prevent saturation. In addition, adult neurogenesis in the hippocampus is proposed to be involved in the down-scaling of synaptic efficacy. Here, we show that adult neurogenesis in male rats plays a crucial role in the maintenance of hippocampal capacity for memory (learning and/or memory formation). Neurogenesis regulated the maintenance of LTP, with decreases and increases in neurogenesis prolonging or shortening LTP persistence, respectively. Artificial saturation of hippocampal LTP impaired memory capacity in contextual fear conditioning, which completely recovered after 14 d, which was the time required for LTP to decay to the basal level. Memory capacity gradually recovered in parallel with neurogenesis-mediated gradual decay of LTP. Ablation of neurogenesis by x-ray irradiation delayed the recovery of memory capacity, whereas enhancement of neurogenesis using a running wheel sped up recovery. Therefore, one benefit of ongoing adult neurogenesis is the maintenance of hippocampal memory capacity through homeostatic renewing of hippocampal memory circuits. Decreased neurogenesis in aged animals may be responsible for the decline in cognitive function with age. SIGNIFICANCE STATEMENT Learning many events each day increases synaptic efficacy via LTP, which can prevent the storage of new memories in the hippocampal circuit. In this study, we demonstrate that hippocampal capacity for the storage of new memories is maintained by ongoing adult neurogenesis through homoeostatic renewing of hippocampal circuits in rats. A decrease or an increase in neurogenesis, respectively, delayed or sped up the recovery of memory capacity, suggesting that hippocampal adult neurogenesis plays a critical role in reducing LTP saturation and keeps the gate open for new memories by clearing out the old memories from the hippocampal memory circuit.

Memory coding strengthens synaptic efficacy in the hippocampus via a long-term potentiation (LTP)-like mechanism. Given that animals are able to store memories of everyday experiences, hippocampal circuits should be able to avoid saturation of overall synaptic weight to preserve learning capacity. However, the underlying mechanism for this is still poorly understood. Here, we show that adult neurogenesis in rats plays a crucial role in the maintenance of the hippocampal learning capacity for learning. Artificial saturation with hippocampal LTP impaired learning capacity in contextual fear conditioning, which then completely recovered after 14 days, when LTP had decayed to the basal level. Ablation of neurogenesis by X-ray irradiation delayed the recovery of learning capacity, while enhancement of neurogenesis using running wheel sped up the recovery. Thus, one benefit of ongoing adult neurogenesis is the maintenance of hippocampal learning capacity through homeostatic renewing of hippocampal memory circuits. Decreased neurogenesis in aged animals may underlie declines in cognitive function with aging.

Unrelated memories get blurred together If one retrieves two memories around the same time, a small number of neurons will become involved in both memories. Yokose et al. investigated the cellular ensemble mechanisms underlying the association between two such memories. In mice, a small population of neurons mediates the association. Memory traces for two independent emotional memories in the brain partially overlapped when the two memories were retrieved synchronously to create a linkage. Suppressing the activity of the overlapping memory trace interrupted the linkage without damaging the original memories. Science , this issue p. 398

The brain stores and recalls memories through a set of neurons, termed engram cells. However, it is unclear how these cells are organized to constitute a corresponding memory trace. We established a unique imaging system that combines Ca2+ imaging and engram identification to extract the characteristics of engram activity by visualizing and discriminating between engram and non-engram cells. Here, we show that engram cells detected in the hippocampus display higher repetitive activity than non-engram cells during novel context learning. The total activity pattern of the engram cells during learning is stable across post-learning memory processing. Within a single engram population, we detected several sub-ensembles composed of neurons collectively activated during learning. Some sub-ensembles preferentially reappear during post-learning sleep, and these replayed sub-ensembles are more likely to be reactivated during retrieval. These results indicate that sub-ensembles represent distinct pieces of information, which are then orchestrated to constitute an entire memory., The brain stores memories through a set of neurons known as engram cells. Here, the authors show that engram cells in the mouse hippocampus are organized into sub-ensembles representing distinct pieces of information, which are then orchestrated to constitute an entire memory.

The regulation of microtubule dynamics is important for the appropriate arborization of neuronal dendrites during development, which in turn is critical for the formation of functional neural networks. Here we show that stathmin, a microtubule destabilizing factor, is downregulated at both the expression and activity levels during cerebellar development, and this down-regulation contributes to dendritic arborization. Stathmin overexpression drastically limited the dendritic growth of cultured Purkinje cells. The stathmin activity was suppressed by neural activity and CaMKII-dependent phosphorylation at Ser16, which led to dendritic arborization. Stathmin phosphorylation at Ser16 was mediated by the activation of voltage-gated calcium channels and metabotropic glutamate receptor 1. Although overexpression of SCG10, a member of the stathmin family, also limited the dendritic arborization, SCG10 did not mediate the CaMKII regulation of dendritic development. These results suggest that calcium elevation activates CaMKII, which in turn phosphorylates stathmin at Ser16 to stabilize dendritic microtubules. siRNA knockdown of endogenous stathmin significantly reduced dendritic growth in Purkinje cells. Thus, these data suggest that proper regulation of stathmin activity is a key factor for controlling the dendritic microtubule dynamics that are important for neuronal development.

Activity-dependent synaptic plasticity has been thought to be a cellular basis of memory and learning. The late phase of long-term potentiation (L-LTP), distinct from the early phase, lasts for up to 6 h and requires de novo synthesis of mRNA and protein. Many LTP-related genes are enhanced in the hippocampus during pentyrenetetrazol (PTZ)- and kainate (KA)-mediated neural activation. In this study, mice were administered intraperitoneal injections of PTZ 10 times, once every 48 h, and showed an increase in seizure indexes. Genes related to plasticity were efficiently induced in the mouse hippocampus. We used a PCR-based cDNA subtraction method to isolate genes that are expressed in the hippocampus of repeatedly PTZ-treated mice. One of these genes, neural activity-related RING finger protein (NARF), encodes a new protein containing a RING finger, B-box zinc finger, coiled-coil (RBCC domain) and β-propeller (NHL) domain, and is predominantly expressed in the brain, especially in the hippocampus. In addition, KA up-regulated the expression of NARF mRNA in the hippocampus. This increase correlated with the activity of the NMDA receptor. By analysis using GFP-fused NARF, the protein was found to localize in the cytoplasm. Enhanced green fluorescent protein-fused NARF was also localized in the neurites and growth cones in neuronal differentiated P19 cells. The C-terminal β-propeller domain of NARF interacts with myosin V, which is one of the most abundant myosin isoforms in neurons. The NARF protein increases in hippocampal and cerebellar neurons after PTZ-induced seizure. These observations indicated that NARF expression is enhanced by seizure-related neural activities, and NARF may contribute to the alteration of neural cellular mechanisms along with myosin V.

In the mammalian central nervous system (CNS), transcription factor activator protein 2 (AP-2) is one of the critical regulatory factors for neural gene expression and neural development. As AP-2 has diverged into several subtypes, i.e. AP-2alpha, -2beta, and 2.2, we investigated the distribution of the AP-2 subtypes in the adult mouse brain by in situ hybridization using subtype-specific probes. Though AP-2 was essentially expressed in most regions of the brain, the hippocampus and cerebellum Purkinje cells exhibited a relatively high concentration of transcripts of any of the AP-2 subtypes. Among AP-2alpha variants, the expression of variant 1 was considerably lower than that of variant 3. Hence, the expression pattern of AP-2alpha variant 3 is suggested to represent the major gene expression of AP-2alpha. On the other hand, the expression of AP-2beta messenger RNA (mRNA) was higher than that of AP-2alpha in many regions. Especially, the olfactory bulb, hippocampus, cerebellum, and cerebral cortex contained an abundance of these mRNAs. Different from those of AP-2alpha, AP-2beta mRNAs were detected in considerable amounts in the glanular cells as well as in Purkinje cells. AP-2.2 gene expression was weak throughout the brain. Consequently, we found that various AP-2 subtypes and variants were expressed in a similar distribution pattern with each having its own specific intensity but that their precise distribution profiles were not exactly the same. In the mature brain, AP-2 is thought to regulate neural gene expression through specific and redundant association with a target gene.

Many studies have reported the roles played by regulated proteolysis in synaptic plasticity and memory, but the role of autophagy in neurons remains unclear. In mammalian cells, autophagy functions in the clearance of long-lived proteins and organelles and in adaptation to starvation. In neurons, although autophagy-related proteins (ATGs) are highly expressed, autophagic activity markers, autophagosome (AP) number, and light chain protein 3-II (LC3-II) are low compared with other cell types. In contrast, conditional knock-out of ATG5 or ATG7 in mouse brain causes neurodegeneration and behavioral deficits. Therefore, this study aimed to test whether autophagy is especially regulated in neurons to adapt to brain functions. In cultured rat hippocampal neurons, we found that KCl depolarization transiently increased LC3-II and AP number, which was partially inhibited with APV, an NMDA receptor (NMDAR) inhibitor. Brief low-dose NMDA, a model of chemical long-term depression (chem-LTD), increased LC3-II with a time course coincident with Akt and mammalian target of rapamycin (mTOR) dephosphorylation and degradation of GluR1, an AMPA receptor (AMPAR) subunit. Downstream of NMDAR, the protein phosphatase 1 inhibitor okadaic acid, PTEN inhibitor bpV(HOpic), autophagy inhibitor wortmannin, and short hairpin RNA-mediated knockdown of ATG7 blocked chem-LTD-induced autophagy and partially recovered GluR1 levels. After chem-LTD, GFP-LC3 puncta increased in spines and in dendrites when AP-lysosome fusion was blocked. These results indicate that neuronal stimulation induces NMDAR-dependent autophagy through PI3K-Akt-mTOR pathway inhibition, which may function in AMPAR degradation, thus suggesting autophagy as a contributor to NMDAR-dependent synaptic plasticity and brain functions.

ARD1 and NAT1 constitute an N-acetyltransferase complex where ARD1 holds the enzymatic activity of the complex. The ARD1-NAT1 complex mediates N-terminal acetylation of nascent polypeptides that emerge from ribosomes after translation. ARD1 may also acetylate the internal lysine residues of proteins. Although ARD1 and NAT1 have been found in the brain, the physiological role and substrates of the ARD1-NAT1 complex in neurons remain unclear. Here we investigated role of N-acetyltransferase activity in the process of neuronal development. Expression of ARD1 and NAT1 increased during dendritic development, and both proteins colocalized with microtubules in dendrites. The ARD1-NAT1 complex displayed acetyltransferase activity against a purified microtubule fraction in vitro. Inhibition of the complex limited the dendritic extension of cultured neurons. These findings suggest that the ARD1-NAT1 complex has acetyltransferase activity against microtubules in dendrites. Regulation by acetyltransferase activity is a novel mechanism that is required for dendritic arborization during neuronal development.

The proper regulation of microtubule (MT) structure is important for dendritic and neural circuit development. However, the relationship between the regulation of the MTs in dendrites and the formation of neural function is still unclear. Stathmin is a MT destabilizer, and we have previously reported that the expression and the activity of stathmin is downregulated during cerebellar Purkinje cell (PC) development. In this study, we generated transgenic mice that specifically overexpress the constitutively active form of stathmin in the PCs. These mutant mice did not show any obvious morphological or excitatory transmission abnormalities in the cerebellum. In contrast, we observed a decline in the expression of MAP2 and KIF5 signal in the PC dendrites and a discoordination of motor function in the mutant mice, although they displayed normal general behavior. These data indicate that the overexpression of stathmin disrupts dendritic MT organization, motor protein distribution, and neural function in PCs.

Using a partial cDNA sequence and a 5'-RACE technique, we isolated a novel cDNA from rat liver referred to as DB83. DB83 had four hydrophobic trans-membrane domains and one N-myristoylation site as well as multiple possible phosphorylation sites. The db83 gene was highly expressed in the liver and significantly in brain, lungs and kidneys. We suggest that DB83 is a tissue-specific putative membrane protein. p6

NeuroD-related factor (NDRF) is a basic helix-loop- helix (bHLH) protein whose expression is restricted to the central nervous system, and is considered to be responsible for maintenance of differentiated neurons as well as neurogenesis. NDRF structurally resembles NeuroD in the bHLH region and can induce neurogenesis ectopically in ectodermal cells of the Xenopus embryo. In this study, we delineated the functional domains of NDRF. Using GAL4/NDRF fusion proteins, we identified the C-terminal activa- tion domain (C-AD) in NDRF between amino acid positions 294 and 383. This region was highly homol- ogous to one part of the activation domain of NeuroD. We further investigated the transactivational function of C-AD in the mouse type 1 inositol 1,4,5- trisphosphate receptor promoter, which has an NDRF site. Truncation of C-AD resulted in reduction of the activation function, whereas the DNA-binding specificity was not affected. These results suggest that C-AD has a stimulatory function in the mammalian nervous system.

The type 1 inositol 1,4,5-trisphosphate receptor (IP 3 R1) is a Ca 2 + channel protein that is expressed abundantly in the CNS, such as in the cerebellar Purkinje cells and hippocampus. We previously demonstrated that the box-I element, which is located -334 relative to the transcription initiation site of the mouse IP 3 R1 gene and includes an E-box consensus sequence, is involved in the up-regulation of such IP 3 R1 gene expression. Furthermore, the previous study also indicated that some CNS-related basic helix-loop-helix (bHLH) factors bind to the box-I and activate IP 3 R1 gene expression. In this study, we demonstrated that one of the CNS-related bHLH factors, neuronal differentiation factor (NeuroD)-related factor (NDRF), specifically bound to the box-I sequence with a ubiquitously expressed bHLH protein, E47, and activated IP 3 R1 gene expression. In situ hybridization of adult mouse brain revealed that IP 3 R1 and NDRF mRNA were co-expressed in many subsets of neurons, highly in Purkinje cells and hippocampus and moderately in cerebral cortex, olfactory bulb, and caudate putamen. Furthermore, the spatiotemporal expression patterns of these two genes resembled one another throughout postnatal development of the mouse CNS. From these results, we suggest that NDRF is involved in the tissue-specific regulation of IP 3 R1 gene expression in the CNS.