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Serotonergic neurons originating from the raphe nuclei have been proposed to regulate corticotropin‐releasing factor (CRF) neurons in the paraventricular nucleus of the hypothalamus (PVH). Since glutamate‐ and γ‐aminobutyric acid (GABA)‐containing neurons, constituting the hypothalamic local circuits, innervate PVH CRF neurons, we examined whether they mediate the actions of serotonin (5‐hydroxytryptamine [5‐HT]) on CRF neurons. Spontaneous excitatory postsynaptic currents (sEPSCs) or spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded in PVH CRF neurons, under whole cell patch‐clamp, using the CRF‐modified yellow fluorescent protein (Venus) ΔNeo mouse. Serotonin elicited an increase in the frequency of sEPSCs in 77% of the cells and a decrease in the frequency of sIPSCs in 71% of the cells, tested in normal medium. Neither the amplitude nor decay time of sEPSC and sIPSC was affected, thus the site(s) of action of serotonin may be presynaptic. In the presence of tetrodotoxin (TTX), serotonin had no significant effects on either parameter of sEPSC or sIPSC, indicating that the effects of serotonin are action potential‐dependent, and that the presynaptic interneurons are largely intact within the slice; distant neurons may exist, though, since some 20%–30% of neurons did not respond to serotonin without TTX. We next examined through what receptor subtype(s) serotonin exerts its effects on presynaptic interneurons. DOI (5‐HT2A/2C agonist) mimicked the action of serotonin on the sIPSCs, and the serotonin‐induced decrease in sIPSC frequency was inhibited by a selective 5‐HT2C antagonist RS102221. 8‐OH‐DPAT (5‐HT1A/7 agonist) mimicked the action of serotonin on the sEPSCs, and the serotonin‐induced increase in sEPSC frequency was inhibited by a selective 5‐HT7 antagonist SB269970. Thus, serotonin showed a dual action on PVH CRF neurons, by upregulating glutamatergic‐ and downregulating GABAergic interneurons; the former may partly be mediated by 5‐HT7 receptors, whereas the latter by 5‐HT2C receptors. The CRF‐Venus ΔNeo mouse was useful for the electrophysiological examination. [ABSTRACT FROM AUTHOR]

NRF2 is a transcription factor responsible for antioxidant stress responses that is usually regulated in a redox‐dependent manner. p62 bodies formed by liquid–liquid phase separation contain Ser349‐phosphorylated p62, which participates in the redox‐independent activation of NRF2. However, the regulatory mechanism and physiological significance of p62 phosphorylation remain unclear. Here, we identify ULK1 as a kinase responsible for the phosphorylation of p62. ULK1 colocalizes with p62 bodies, directly interacting with p62. ULK1‐dependent phosphorylation of p62 allows KEAP1 to be retained within p62 bodies, thus activating NRF2. p62S351E/+ mice are phosphomimetic knock‐in mice in which Ser351, corresponding to human Ser349, is replaced by Glu. These mice, but not their phosphodefective p62S351A/S351A counterparts, exhibit NRF2 hyperactivation and growth retardation. This retardation is caused by malnutrition and dehydration due to obstruction of the esophagus and forestomach secondary to hyperkeratosis, a phenotype also observed in systemic Keap1‐knockout mice. Our results expand our understanding of the physiological importance of the redox‐independent NRF2 activation pathway and provide new insights into the role of phase separation in this process. Synopsis: NRF2 is a master transcription factor for antioxidant stress that is usually regulated by redox states of KEAP1. This study shows that when ULK1 phosphorylates phase‐separated p62 bodies, they bind KEAP1, leading to NRF2‐activation in a redox‐independent manner. ULK1 phosphorylates p62 bodies formed by liquid–liquid phase separation.KEAP1 is retained into these phosphorylated p62 bodies, leading to NRF2 activation.Persistent activation of NRF2 by the redox‐independent pathway causes hyperkeratosis in vivo.The resulting esophageal and forestomach obstruction causes severe growth retardation due to malnutrition. [ABSTRACT FROM AUTHOR]

Extended pluripotent stem cells (EPSCs) derived from mice and humans showed an enhanced potential for chimeric formation. By exploiting transcriptomic approaches, we assessed the differences in gene expression profile between extended EPSCs derived from mice and humans, and those newly derived from the common marmoset (marmoset; Callithrix jacchus). Although the marmoset EPSC‐like cells displayed a unique colony morphology distinct from murine and human EPSCs, they displayed a pluripotent state akin to embryonic stem cells (ESCs), as confirmed by gene expression and immunocytochemical analyses of pluripotency markers and three‐germ‐layer differentiation assay. Importantly, the marmoset EPSC‐like cells showed interspecies chimeric contribution to mouse embryos, such as E6.5 blastocysts in vitro and E6.5 epiblasts in vivo in mouse development. Also, we discovered that the perturbation of gene expression of the marmoset EPSC‐like cells from the original ESCs resembled that of human EPSCs. Taken together, our multiple analyses evaluated the efficacy of the method for the derivation of marmoset EPSCs. [ABSTRACT FROM AUTHOR]

The semaphorin family is a well‐characterized family of secreted or membrane‐bound proteins that are involved in activity‐independent neurodevelopmental processes, such as axon guidance, cell migration, and immune functions. Although semaphorins have recently been demonstrated to regulate activity‐dependent synaptic scaling, their roles in Hebbian synaptic plasticity as well as learning and memory remain poorly understood. Here, using a rodent model, we found that an inhibitory avoidance task, a hippocampus‐dependent contextual learning paradigm, increased secretion of semaphorin 3A in the hippocampus. Furthermore, the secreted semaphorin 3A in the hippocampus mediated contextual memory formation likely by driving AMPA receptors into hippocampal synapses via the neuropilin1–plexin A4–semaphorin receptor complex. This signaling process involves alteration of the phosphorylation status of collapsin response mediator protein 2, which has been characterized as a downstream molecule in semaphorin signaling. These findings implicate semaphorin family as a regulator of Hebbian synaptic plasticity and learning. [ABSTRACT FROM AUTHOR]

The properties of microglia largely differ depending on aging as well as on brain regions. However, there are few studies that investigated the functional importance of such heterogeneous properties of microglia at the molecular level. Voltage‐gated proton channel, Hv1/VSOP, could be one of the candidates which confers functional heterogeneity among microglia since it regulates brain oxidative stress in age‐dependent manner. In this study, we found that Hv1/VSOP shows brain region‐dependent heterogeneity of gene expression with the highest level in the striatum. We studied the importance of Hv1/VSOP in two different brain regions, the cerebral cortex and striatum, and examined their relationship with aging (using mice of different ages). In the cortex, we observed the age‐dependent impact of Hv1/VSOP on oxidative stress, microglial morphology, and gene expression profile. On the other hand, we found that the age‐dependent significance of Hv1/VSOP was less obvious in the striatum than the cortex. Finally, we performed a battery of behavioral experiments on Hv1/VSOP‐deficient mice both at young and aged stages to examine the effect of aging on Hv1/VSOP function. Hv1/VSOP‐deficient mice specifically showed a marked difference in behavior in light/dark transition test only at aged stages, indicating that anxiety state is altered in aged Hv1/VSOP mice. This study suggests that a combination of brain region heterogeneity and animal aging underscores the functional importance of Hv1/VSOP in microglia. [ABSTRACT FROM AUTHOR]

Aims: Adult hippocampal neurogenesis plays an important role in neuronal plasticity and maintenance in mammals. Low‐threshold voltage‐gated T‐type calcium channels produce calcium spikes that increase fast action potentials in newborn cells in the hippocampal dentate gyrus (DG); however, their role in adult hippocampal neurogenesis remains unclear. Here, we demonstrate impaired adult hippocampal neurogenesis in Cav3.1T‐type calcium channel knockout mice. Methods and Results: Cav3.1T‐type calcium channel was predominantly localized in neuronal progenitor cells of the mouse hippocampal DG. By counting the number of 5‐bromo‐2′‐deoxyuridine‐labeled cells, decreased proliferation and survival of newly generated cells were observed in the adult hippocampal DG in Cav3.1 knockout mice as compared to wild‐type (WT) mice. Moreover, the degree of maturation of doublecortin‐positive cells in Cav3.1 knockout mice was lower than that in WT mice, suggesting that Cav3.1 deletion may impair neuronal differentiation. Consistent with impaired hippocampal neurogenesis, Cav3.1 knockout mice showed decreased social interaction. Reduced phosphorylation levels of calcium/calmodulin‐dependent protein kinase II and protein kinase B were closely associated with impaired hippocampal neurogenesis in Cav3.1 knockout mice. Moreover, the mRNA and protein expression levels of brain‐derived neurotrophic factor, important for neurogenesis, were significantly decreased in Cav3.1 knockout mice. Finally, gene ontology analysis revealed alterations in genes related to the promotion of cell death/apoptosis and suppression of cell proliferation/neuronal differentiation pathways, including Bdnf. Conclusion: These results suggest that the Cav3.1T‐type calcium channel may be a key molecule required for cell proliferation, survival and neuronal differentiation in newly generated cells of the adult mouse hippocampus. [ABSTRACT FROM AUTHOR]

Aim: Sphingosine‐1‐phosphate (S1P) and ceramide are bioactive sphingolipids known to be important in regulating numerous processes involved in cancer progression. The aim of this study was to determine the absolute levels of sphingolipids in hepatocellular carcinoma (HCC) utilizing data obtained from surgical specimens. In addition, we explored the clinical significance of S1P in patients with HCC and the biological role of S1P in HCC cells. Methods: Tumors and normal liver tissues were collected from 20 patients with HCC, and sphingolipids were measured by mass spectrometry. The Cancer Genome Atlas (TCGA) cohort was utilized to evaluate gene expression of enzymes related to sphingolipid metabolism. Immunohistochemistry of phospho‐sphingosine kinase 1 (SphK1), an S1P‐producing enzyme, was performed for 61 surgical specimens. CRISPR/Cas9‐mediated SphK1 knockout cells were used to examine HCC cell biology. Results: S1P levels were substantially higher in HCC tissue compared with normal liver tissue. Levels of other sphingolipids upstream of S1P in the metabolic cascade, such as sphingomyelin, monohexosylceramide and ceramide, were also considerably higher in HCC tissue. Enzymes involved in generating S1P and its precursor, ceramide, were found in higher levels in HCC compared with normal liver tissue. Immunohistochemical analysis found that phospho‐SphK1 expression was associated with tumor size. Finally, in vitro assays indicated that S1P is involved in the aggressiveness of HCC cells. Conclusions: Sphingolipid levels, including S1P and ceramide, were elevated in HCC compared with surrounding normal liver tissue. Our findings suggest S1P plays an important role in HCC tumor progression, and further examination is warranted. [ABSTRACT FROM AUTHOR]

Key points: Some ion channels are known to behave as inductors and make up the parallel resonant circuit in the plasma membrane of neurons, which enables neurons to respond to current inputs with a specific frequency (so‐called 'resonant properties').Here, we report that heterologous expression of mouse Kv11 voltage‐dependent K+ channels generate resonance and oscillation at depolarized membrane potentials in HEK293 cells; expressions of individual Kv11 subtypes generate resonance and oscillation with different frequency properties.Kv11.3‐expressing HEK293 cells exhibited transient conductance changes that opposed the current changes induced by voltage steps; this probably enables Kv11 channels to behave like an inductor.The resonance and oscillation of inferior olivary neurons were impaired at the resting membrane potential in Kv11.3 knockout mice.This study helps to elucidate basic ion channel properties that are crucial for the frequency responses of neurons. The plasma membranes of some neurons preferentially respond to current inputs with a specific frequency, and output as large voltage changes. This property is called resonance, and is thought to be mediated by ion channels that show inductor‐like behaviour. However, details of the candidate ion channels remain unclear. In this study, we mainly focused on the functional roles of Kv11 potassium (K+) channels, encoded by ether‐á‐go‐go‐related genes, in resonance in mouse inferior olivary (IO) neurons. We transfected HEK293 cells with long or short splice variants of Kv11.1 (Merg1a and Merg1b) or Kv11.3, and examined membrane properties using whole‐cell recording. Transfection with Kv11 channels reproduced resonance at membrane potentials depolarized from the resting state. Frequency ranges of Kv11.3‐, Kv11.1(Merg1b)‐ and Kv11.1(Merg1a)‐expressing cells were 2–6 Hz, 2–4 Hz, and 0.6–0.8 Hz, respectively. Responses of Kv11.3 currents to step voltage changes were essentially similar to those of inductor currents in the resistor–inductor–capacitor circuit. Furthermore, Kv11 transfections generated membrane potential oscillations. We also confirmed the contribution of HCN1 channels as a major mediator of resonance at more hyperpolarized potentials by transfection into HEK293 cells. The Kv11 current kinetics and properties of Kv11‐dependent resonance suggested that Kv11.3 mediated resonance in IO neurons. This finding was confirmed by the impairment of resonance and oscillation at –30 to –60 mV in Kcnh7 (Kv11.3) knockout mice. These results suggest that Kv11 channels have important roles in inducing frequency‐dependent responses in a subtype‐dependent manner from resting to depolarized membrane potentials. Key points: Some ion channels are known to behave as inductors and make up the parallel resonant circuit in the plasma membrane of neurons, which enables neurons to respond to current inputs with a specific frequency (so‐called 'resonant properties').Here, we report that heterologous expression of mouse Kv11 voltage‐dependent K+ channels generate resonance and oscillation at depolarized membrane potentials in HEK293 cells; expressions of individual Kv11 subtypes generate resonance and oscillation with different frequency properties.Kv11.3‐expressing HEK293 cells exhibited transient conductance changes that opposed the current changes induced by voltage steps; this probably enables Kv11 channels to behave like an inductor.The resonance and oscillation of inferior olivary neurons were impaired at the resting membrane potential in Kv11.3 knockout mice.This study helps to elucidate basic ion channel properties that are crucial for the frequency responses of neurons. [ABSTRACT FROM AUTHOR]

In the cerebellum, GluD2 is exclusively expressed in Purkinje cells, where it regulates synapse formation and regeneration, synaptic plasticity, and motor learning. Delayed cognitive development in humans with GluD2 gene mutations suggests extracerebellar functions of GluD2. However, extracerebellar expression of GluD2 and its relationship with that of GluD1 are poorly understood. GluD2 mRNA and protein were widely detected, with relatively high levels observed in the olfactory glomerular layer, medial prefrontal cortex, cingulate cortex, retrosplenial granular cortex, olfactory tubercle, subiculum, striatum, lateral septum, anterodorsal thalamic nucleus, and arcuate hypothalamic nucleus. These regions were also enriched for GluD1, and many individual neurons coexpressed the two GluDs. In the retrosplenial granular cortex, GluD1 and GluD2 were selectively expressed at PSD‐95‐expressing glutamatergic synapses, and their coexpression on the same synapses was shown by SDS‐digested freeze‐fracture replica labeling. Biochemically, GluD1 and GluD2 formed coimmunoprecipitable complex formation in HEK293T cells and in the cerebral cortex and hippocampus. We further estimated the relative protein amount by quantitative immunoblotting using GluA2/GluD2 and GluA2/GluD1 chimeric proteins as standards for titration of GluD1 and GluD2 antibodies. Intriguingly, the relative amount of GluD2 was almost comparable to that of GluD1 in the postsynaptic density fraction prepared from the cerebral cortex and hippocampus. In contrast, GluD2 was overwhelmingly predominant in the cerebellum. Thus, we have determined the relative extracerebellar expression of GluD1 and GluD2 at regional, neuronal, and synaptic levels. These data provide a molecular–anatomical basis for possible competitive and cooperative interactions of GluD family members at synapses in various brain regions. [ABSTRACT FROM AUTHOR]

Oligodendrocytes (OLs) are myelinating cells of the central nervous system. Recent studies have shown that mechanical factors influence various cell properties. Mechanical stimulation can be transduced into intracellular biochemical signals through mechanosensors, such as integrin, p130Cas, talin and vinculin. However, the molecular mechanisms underlying the mechanical regulation of OLs by mechanosensors remain largely unknown. We found that morphology of OL was affected by knockdown of the mechanosensors p130Cas or talin1. Stretching of OL precursor cells induced the phosphorylation of p130Cas and talin‐associated assembly of vinculin. Shear stress decreased the number of OL processes, whereas these effects were mechanically suppressed by dominant‐negative (DN) p130Cas, but not by DN‐talin1. To investigate the roles of p130Cas in post‐natal OLs in vivo, we constructed a novel p130Cas knock‐in mouse and found overexpression of p130Cas in vivo affected the number of mature OLs in the cortex. These results indicate that the mechanosensor p130Cas controls both OL morphogenesis and maturation. [ABSTRACT FROM AUTHOR]

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. Compared with wild‐type mouse (top), AMPA receptor GluA1 Cys811Ser palmitoylation‐deficient mouse (bottom) shows abnormal expression pattern of immediate early gene Arc in the hippocampal dentate gyrus. 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. [ABSTRACT FROM AUTHOR]

Multiple sclerosis (MS) is an autoimmune disease characterized by immune‐mediated inflammation, which attacks the myelin sheath. MS pursues a relapsing and remitting course with varying intervals between symptoms. The main clinical pathological features include inflammation, myelin sheath destruction and plaque formation in the central nervous system (CNS). We previously reported that cystatin F (CysF) expression is induced in demyelinating lesions that are accompanied by active remyelination (referred to as shadow plaques) but is down‐regulated in chronic demyelinated lesions (plaques) in the spinal cord of MS patients and in several murine models of demyelinating disease. CysF is a cathepsin protease inhibitor whose major target is cathepsin C (CatC), which is co‐expressed in demyelinating regions in Plp4e/− mice, a model of chronic demyelination. Here, we report the time course of CatC and CysF expression and describe the symptoms in a mouse experimental autoimmune encephalomyelitis (EAE) model using CatC knockdown (KD) and CatC over‐expression (OE) mice. In myelin oligodendrocyte glycoprotein (MOG)‐EAE, CatC positive cells were found to infiltrate the CNS at an early stage prior to any clinical signs, in comparison to WT mice. CysF expression was not observed at this early stage, but appeared later within shadow plaques. CatC expression was found in chronic demyelinated lesions but was not associated with CysF expression, and CatCKD EAE mouse showed delayed demyelination. Whereas, CatCOE in microglia significantly increased severity of demyelination in the MOG‐EAE model. Thus, these results demonstrate that CatC plays a major role in MOG‐EAE. Cathepsin C (CatC) plays a major role in autoimmune induced demyelination, MOG‐EAE model: CatC is expressed in microglia and neutrophil at an early demyelination. Modulation of CatC level in microglia affected in severity of demyelination. CatC and Cystatin F (CysF) interaction play an important role in both pathogenesis and progress of inflammatory demyelination. The finding in this study might be important for developing new therapeutic approaches to demyelinating diseases. [ABSTRACT FROM AUTHOR]

Many inflammatory mediators are involved in the process of carcinogenesis and cancer progression. In addition to cytokines and chemokines, lipid mediators have recently attracted attention as signaling molecules associated with inflammatory diseases. Sphingosine‐1‐phosphate (S1P) is a pleiotropic lipid mediator that regulates cell survival and migration, immune cell recruitment, angiogenesis and lymphangiogenesis. S1P also plays a significant role in inflammation and cancer. The gradation of S1P concentration in the blood, lymph and tissue regulates lymphocyte trafficking, an important component of inflammation. Furthermore, cancer cells produce elevated levels of S1P, contributing to the tumor microenvironment and linking cancer and inflammation. Future technological advances may reveal greater detail about the mechanisms of S1P regulation in the tumor microenvironment and the contribution of S1P to cancer progression. Considering the critical role of S1P in linking inflammation and cancer, it is possible that the S1P signaling pathway could be a novel therapeutic target for cancers with chronic inflammation. Sphingosine‐1‐phosphate (S1P) is a pleiotropic lipid mediator that regulates cell survival and migration, immune cell recruitment, angiogenesis and lymphangiogenesis. Cancer cells produce elevated levels of S1P, contributing to the tumor microenvironment and linking cancer and inflammation. Considering the critical role of S1P in linking inflammation and cancer, it is possible that the S1P signaling pathway could be a novel therapeutic target for cancers with chronic inflammation. [ABSTRACT FROM AUTHOR]

Purkinje cell degeneration (pcd) was first identified in a spontaneous mouse mutant showing cerebellar ataxia. In addition to cerebellar Purkinje cells (PCs), retinal photoreceptors, mitral cells in the olfactory bulb, and a discrete subpopulation of thalamic neurons also degenerate in the mutant brains. The gene responsible for the pcd mutant is Nna1, also known as ATP/GTP binding protein 1 or cytosolic carboxypeptidase‐like 1, which encodes a zinc carboxypeptidase protein. To investigate pathogenesis of the pcd mutation in detail, we generated a conditional Nna1 allele targeting the carboxypeptidase domain at C‐terminus. After Cre recombination and heterozygous crossing, we generated Nna1 knockout (KO) mice and found that the Nna1 KO mice began to show cerebellar ataxia at postnatal day 20 (P20). Most PCs degenerated until 4‐week‐old, except lobule X. Activated microglia and astrocytes were also observed in the Nna1 KO cerebellum. In the mutant brain, the Nna1 mRNA level was dramatically reduced, suggesting that nonsense‐mediated mRNA decay occurs in it. Since the Nna1 protein acts as a de‐glutamatase on the C‐terminus of α‐tubulin and β‐tubulin, increased polyglutamylated tubulin was detected in the Nna1 KO cerebellum. In addition, the endoplasmic reticulum stress marker, C/EBP homologous protein (CHOP), was up‐regulated in the mutant PCs. We report the generation of a functional Nna1 conditional allele and possible mechanisms of PC death in the Nna1 KO in the cerebellum. Open Practices: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. A novel Nna1 flox allele was generated, which is causative gene for Purkinje cell degeneration (pcd) mutant mice. The Nna1 null mice showed pcd phenotype, as well as neurodegeneration in the retina and olfactory bulb and defect in spermatogenesis. In addition, Calbindin D‐28k immunostaining at 4‐week‐old showed that Purkinje cells of the lobule X are relatively resistant to neuronal cell death. This Nna1 flox allele is useful for analyses of Nna1 functions in multiple organs (Scale bars: 500 μm). Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/ [ABSTRACT FROM AUTHOR]

Mammalian eukaryotic translation initiation factor 3 (eIF3) is the largest complex of the translation initiation factors. The eIF3 complex is comprised of thirteen subunits, which are named eIF3a to eIF3 m in most multicellular organisms. The eIF3e gene locus is one of the most frequent integration sites of mouse mammary tumor virus (MMTV), which induces mammary tumors in mice. MMTV‐integration events result in the expression of C‐terminal‐truncated eIF3e proteins, leading to mammary tumor formation. We have shown that tumor formation can be partly caused by activation of hypoxia‐inducible factor 2α. To investigate the function of eIF3e in mammals, we generated eIF3e‐deficient mice. These eIF3e−/− mice are embryonically lethal, while eIF3e+/− mice are much smaller than wild‐type mice. In addition, eIF3e+/− mouse embryonic fibroblasts (MEFs) contained reduced levels of eIF3a and eIF3c subunits and exhibited reduced cellular proliferation. These results suggest that eIF3e is essential for embryonic development in mice and plays a role in maintaining eIF3 integrity. [ABSTRACT FROM AUTHOR]

F-actin-binding protein drebrin has two major isoforms: drebrin A and drebrin E. Drebrin A is the major isoform in the adult brain and is highly concentrated in dendritic spines, regulating spine morphology and synaptic plasticity. Conversely, drebrin E is the major isoform in the embryonic brain and regulates neuronal morphological differentiation, but it is also expressed in neurogenic regions of the adult brain. The subventricular zone ( SVZ) is one of the brain regions where adult neurogenesis occurs. Neuroblasts migrate to the olfactory bulb ( OB) and integrate into existing neuronal networks, after which drebrin expression changes from E to A, suggesting that drebrin E plays a specific role in neuroblasts in the adult brain. Therefore, to understand the role of drebrin E in the adult brain, we immunohistochemically analyzed adult neurogenesis using drebrin-null-mutant ( DXKO) mice. In DXKO mice, the number of neuroblasts and cell proliferation decreased, although cell death remained unchanged. These results suggest that drebrin E regulates cell proliferation in the adult SVZ. Surprisingly, the decreased number of neuroblasts in the SVZ did not result in less neurons in the OB. This was because the survival rate of newly generated neurons in the OB increased in DXKO mice. Additionally, when neuroblasts reached the OB, the change in the migratory pathway from tangential to radial was partly disturbed in DXKO mice. These results suggest that drebrin E is involved in a chain migration of neuroblasts. [ABSTRACT FROM AUTHOR]

It has been established that voltage-gated proton channels ( VSOP/Hv1), encoded by Hvcn1, support reactive oxygen species ( ROS) production in phagocytic activities of neutrophils (El Chemaly et al. ) and antibody production in B lymphocytes (Capasso et al. ). VSOP/Hv1 is a potential therapeutic target for brain ischemia, since Hvcn1 deficiency reduces microglial ROS production and protects brain from neuronal damage (Wu et al. ). In the present study, we report that VSOP/Hv1 has paradoxical suppressive role in ROS production in microglia. Extracellular ROS production was lower in neutrophils of Hvcn1−/− mice than WT mice as reported. In contrast, it was drastically enhanced in isolated Hvcn1−/− microglia as compared with cells from WT mice. Actin dynamics was altered in Hvcn1−/− microglia and intracellular distribution of cytosolic NADPH oxidase subunit, p67, was changed. When expression levels of oxidative stress responsive antioxidant genes were compared between WT and Hvcn1−/− in cerebral cortex at different ages of animals, they were slightly decreased in Hvcn1−/− mice at younger stage (1 day, 5 days, 3 weeks old), but drastically increased at aged stage (6 months old), suggesting that the regulation of microglial ROS production by VSOP/Hv1 is age-dependent. We also performed brain ischemic stroke experiments and found that the neuroprotective effect of VSOP/Hv1deficiency on infarct volume depended on the age of animals. Taken together, regulation of ROS production by VSOP/Hv1 is more complex than previously thought and significance of VSOP/Hv1 in microglial ROS production depends on age. [ABSTRACT FROM AUTHOR]

PKN2, a member of the protein kinase N ( PKN) family, has been suggested by in vitro culture cell experiments to bind to Rho/Rac GTPases and contributes to cell-cell contact and cell migration. To unravel the in vivo physiological function of PKN2, we targeted the PKN2 gene. Constitutive disruption of the mouse PKN2 gene resulted in growth retardation and lethality before embryonic day (E) 10.5. PKN2−/− embryo did not undergo axial turning and showed insufficient closure of the neural tube. Mouse embryonic fibroblasts ( MEFs) derived from PKN2−/− embryos at E9.5 failed to grow. Cre-mediated ablation of PKN2 in PKN2flox/flox MEFs obtained from E14.5 embryos showed impaired cell proliferation, and cell cycle analysis of these MEFs showed a decrease in S-phase population. Our results show that PKN2 is essential for mouse embryonic development and cell-autonomous proliferation of primary MEFs in culture. Comparison of the PKN2−/− phenotype with the phenotypes of PKN1 and PKN3 knockout strains suggests that PKN2 has distinct nonredundant functions in vivo, despite the structural similarity and evolutionary relationship among the three isoforms. [ABSTRACT FROM AUTHOR]

Key points At the parallel fibre-Purkinje cell glutamatergic synapse, little or no Ca2+ entry takes place through postsynaptic neurotransmitter receptors, although postsynaptic calcium increases are clearly involved in the synaptic plasticity. Postsynaptic voltage-gated Ca2+ channels therefore constitute the sole rapid postsynaptic Ca2+ signalling mechanism, making it essential to understand how they contribute to the synaptic signalling., Using a selective T-type calcium channel antagonist, we describe a T-type component of the EPSC that is activated by the AMPA receptor-mediated depolarization of the spine and thus will contribute to the local calcium dynamics., This component can amount up to 20% of the EPSC, and this fraction is maintained even at the high frequencies sometimes encountered in sensory processing., Modelling based on our biophysical characterization of T-type calcium channels in Purkinje cells suggests that the brief spine EPSCs cause the activated T-type channels to deactivate rather than inactivate, enabling repetitive activation., Abstract In the cerebellum, sensory information is conveyed to Purkinje cells (PC) via the granule cell/parallel fibre (PF) pathway. Plasticity at the PF-PC synapse is considered to be a mechanism of information storage in motor learning. The induction of synaptic plasticity in the cerebellum and elsewhere usually involves intracellular Ca2+ signals. Unusually, postsynaptic Ca2+ signalling in PF-PC spines does not involve ionotropic glutamatergic receptors because postsynaptic NMDA receptors are absent and the AMPA receptors are Ca2+-impermeable; postsynaptic voltage-gated Ca2+ channels therefore constitute the sole rapid Ca2+ signalling mechanism. Low-threshold activated T-type calcium channels are present at the synapse, although their contribution to PF-PC synaptic responses is unknown. Taking advantage of 3,5-dichloro- N-[1-(2,2-dimethyl-tetrahydro-pyran-4-ylmethyl)-4-fluoro-piperidin-4-ylmethyl]-benzamide, a selective T-type channel antagonist, we show in the mouse that inhibition of these channels reduces PF-PC excitatory postsynaptic currents and excitatory postsynaptic potentials by 15-20%. This contribution was preserved during sparse input and repetitive activity. We characterized the biophysical properties of native T-type channels in young animals and modelled their activation during simulated dendritic excitatory postsynaptic potential waveforms. The comparison of modelled and observed synaptic responses suggests that T-type channels only activate in spines that are strongly depolarized by their synaptic input, a process requiring a high spine neck resistance. This brief and local activation ensures that T-type channels rapidly deactivate, thereby limiting inactivation during repetitive synaptic activity. T-type channels are therefore ideally situated to provide synaptic Ca2+ entry at PF-PC spines. [ABSTRACT FROM AUTHOR]

Kainate-type glutamate receptors (KARs) are tetrameric channels assembled from GluK1-5. GluK1-3 are low-affinity subunits that form homomeric and heteromeric KARs, while GluK4 and GluK5 are high-affinity subunits that require co-assembly with GluK1-3 for functional expression. Although the subunit composition is thought to be highly heterogeneous in the brain, the distribution of KAR subunits at the protein level and their relative abundance in given regions of the brain remain largely unknown. In the present study, we titrated C-terminal antibodies to each KAR subunit using chimeric GluA2-GluK fusion proteins, and measured their relative abundance in the P2 and post-synaptic density (PSD) fractions of the adult mouse hippocampus and cerebellum. Analytical western blots showed that GluK2 and GluK3 were the major KAR subunits, with additional expression of GluK5 in the hippocampus and cerebellum. In both regions, GluK4 was very low and GluK1 was below the detection threshold. The relative amount of low-affinity subunits (GluK2 plus GluK3) was several times higher than that of high-affinity subunits (GluK4 plus GluK5) in both regions. Of note, the highest ratio of high-affinity subunits to low-affinity subunits was found in the hippocampal PSD fraction (0.32), suggesting that heteromeric receptors consisting of high- and low-affinity subunits highly accumulate at hippocampal synapses. In comparison, this ratio was decreased to 0.15 in the cerebellar PSD fraction, suggesting that KARs consisting of low-affinity subunits are more prevalent in the cerebellum. Therefore, low-affinity KAR subunits are predominant in the brain, with distinct subunit combinations between the hippocampus and cerebellum. [ABSTRACT FROM AUTHOR]

Ras GTPase-activating proteins negatively regulate the Ras/Erk signaling pathway, thereby playing crucial roles in the proliferation, function, and development of various types of cells. In this study, we identified a novel Ras GTPase-activating proteins protein, RASAL3, which is predominantly expressed in cells of hematopoietic lineages, including NKT, B, and T cells. We established systemic RASAL3-deficient mice, and the mice exhibited a severe decrease in NKT cells in the liver at 8 weeks of age. The treatment of RASAL3-deficient mice with α-GalCer, a specific agonist for NKT cells, induced liver damage, but the level was less severe than that in RASAL3-competent mice, and the attenuated liver damage was accompanied by a reduced production of interleukin-4 and interferon-γ from NKT cells. RASAL3-deficient NKT cells treated with α-GalCer in vitro presented augmented Erk phosphorylation, suggesting that there is dysregulated Ras signaling in the NKT cells of RASAL3-deficient mice. Taken together, these results suggest that RASAL3 plays an important role in the expansion and functions of NKT cells in the liver by negatively regulating Ras/Erk signaling, and might be a therapeutic target for NKT-associated diseases. [ABSTRACT FROM AUTHOR]

Previous studies have implicated the role of Purkinje cells in motor learning and the underlying mechanisms have also been identified in great detail during the last decades. Here we report that cyclin-dependent kinase 5 (Cdk5)/p35 in Purkinje cell also contributes to synaptic plasticity. We previously showed that p35−/− (p35 KO) mice exhibited a subtle abnormality in brain structure and impaired spatial learning and memory. Further behavioral analysis showed that p35 KO mice had a motor coordination defect, suggesting that p35, one of the activators of Cdk5, together with Cdk5 may play an important role in cerebellar motor learning. Therefore, we created Purkinje cell-specific conditional Cdk5/p35 knockout (L7-p35 cKO) mice, analyzed the cerebellar histology and Purkinje cell morphology of these mice, evaluated their performance with balance beam and rota-rod test, and performed electrophysiological recordings to assess long-term synaptic plasticity. Our analyses showed that Purkinje cell-specific deletion of Cdk5/p35 resulted in no changes in Purkinje cell morphology but severely impaired motor coordination. Furthermore, disrupted cerebellar long-term synaptic plasticity was observed at the parallel fiber-Purkinje cell synapse in L7-p35 cKO mice. These results indicate that Cdk5/p35 is required for motor learning and involved in long-term synaptic plasticity. [ABSTRACT FROM AUTHOR]

Monoacylglycerol lipase ( MGL) is a multifunctional serine hydrolase, which terminates anti-nociceptive endocannabinoid signaling and promotes pro-nociceptive prostaglandin signaling. Accordingly, both acute nociception and its sensitization in chronic pain models are prevented by systemic or focal spinal inhibition of MGL activity. Despite its analgesic potential, the neurobiological substrates of beneficial MGL blockade have remained unexplored. Therefore, we examined the regional, cellular and subcellular distribution of MGL in spinal circuits involved in nociceptive processing. All immunohistochemical findings obtained with light, confocal or electron microscopy were validated in MGL-knockout mice. Immunoperoxidase staining revealed a highly concentrated accumulation of MGL in the dorsal horn, especially in superficial layers. Further electron microscopic analysis uncovered that the majority of MGL-immunolabeling is found in axon terminals forming either asymmetric glutamatergic or symmetric γ-aminobutyric acid/glycinergic synapses in laminae I/ IIo. In line with this presynaptic localization, analysis of double-immunofluorescence staining by confocal microscopy showed that MGL colocalizes with neurochemical markers of peptidergic and non-peptidergic nociceptive terminals, and also with markers of local excitatory or inhibitory interneurons. Interestingly, the ratio of MGL-immunolabeling was highest in calcitonin gene-related peptide-positive peptidergic primary afferents, and the staining intensity of nociceptive terminals was significantly reduced in MGL-knockout mice. These observations highlight the spinal nociceptor synapse as a potential anatomical site for the analgesic effects of MGL blockade. Moreover, the presence of MGL in additional terminal types raises the possibility that MGL may play distinct regulatory roles in synaptic endocannabinoid or prostaglandin signaling according to its different cellular locations in the dorsal horn pain circuitry. [ABSTRACT FROM AUTHOR]

Bcl11b is a haploinsufficient tumor suppressor, mutations or deletion of which has been found in 10-16% of T-cell acute lymphoblastic leukemias. Bcl11bKO/+ heterozygous mice are susceptible to thymic lymphomas, a model of T-cell acute lymphoblastic leukemia, when γ-irradiated, and irradiated Bcl11bKO/+ mice generate clonally expanding or premalignant thymocytes before thymic lymphoma development. Cells with radiation-induced DNA damages are assumed to be the cells of origin in tumors; however, which thymocyte is the tumor cell origin remains obscure. In this study we generated Bcl11bflox/+; Lck-Cre and Bcl11bflox/+; CD4-Cre mice; in the former, loss of one Bcl11b allele occurs in thymocytes at the immature CD4− CD8− stage, whereas in the latter the loss occurs in the more differentiated CD4+ CD8+ double-positive stage. We examined clonal expansion and differentiation of thymocytes in mice 60 days after 3 Gy γ-irradiation. Half (9/18) of the thymuses in the Bcl11bflox/+; Lck-Cre group showed limited rearrangement sites at the T-cell receptor-β ( TCRβ) locus, indicating clonal cell expansion, but none in the Bcl11bflox/+; CD4-Cre group did. This indicates that the origin of the premalignant thymocytes is not in double-positive cells but immature thymocytes. Interestingly, those premalignant thymocytes underwent rearrangement at various different sites of the TCRα locus and the majority showed a higher expression of TCRβ and CD8, and more differentiated phenotypes. This suggests the existence of a subpopulation of immature cells within the premalignant cells that is capable of proliferating and continuously producing differentiated thymocytes. [ABSTRACT FROM AUTHOR]

Functional neural circuit formation during postnatal development involves massive elimination of early-formed redundant synapses and strengthening of necessary synaptic connections. In the cerebellum, one-to-one connection from a climbing fibre (CF) to a Purkinje cell (PC) is established through four distinct phases: (1) strengthening of a single CF among multiple CFs in each PC at postnatal age P3-P7 days, (2) translocation of a single strengthened CF to PC dendrites from around P9, (3) early-phase (P7 to around P11) and (4) late-phase (around P12-P17) elimination of weak CF synapses from PC somata. Mice with PC-selective deletion of the P/Q-type voltage-dependent Ca2+ channel (VDCC) exhibit severe defects in strengthening of single CFs, dendritic translocation of single CFs and CF elimination from P7. In contrast, mice with a mutation of a single allele for the GABA synthesizing enzyme GAD67 show selective impairment of CF elimination from P10. Electrophysiological and Ca2+ imaging data suggest that GABAA receptor-mediated inhibition onto PC somata from putative basket cells influences CF-induced Ca2+ transients and regulates elimination of redundant CF synapses from PC somata at P10-P16. Thus, regulation of Ca2+ influx to PCs through VDCCs is crucial for the four phases of CF synapse elimination during postnatal development. [ABSTRACT FROM AUTHOR]

Objectives NMO-IgG/anti-aquaporin 4 antibody (AQP4-Ab) is a specific diagnostic marker for neuromyelitis optica (NMO). Several clinical phenotypes exist in AQP4-Ab-positive patients, such as typical neuromyelitis optica, limited symptoms of myelitis or optic neuritis. The mechanisms responsible for these differences remain poorly understood. We investigated the difference of AQP4-ab binding reactivity among clinical subtypes of AQP4-Ab-posive NMO cases using 3 kinds of AQP4 proteins. Methods Indirect immunofluorescence staining was utilized to measure the binding intensities of AQP4-Ab to each of the AQP4-transfected HEK 293 cells, including human, mouse, and rat AQP4 (hAQP4, mAQP4, and rAQP4), each has one amino acid substitution at the extracellular domain of AQP4 protein and is speculated to show some different characters as the antigen epitope. Serum samples from 24 AQP4-Ab positive patients, including six patients each from three adult onset groups and child-onset group (group C), were collected. The adult-onset patients were assigned to groups: NMO - typical neuromyelitis optica; ON - relapsing optic neuritis; M - relapsing myelitis. Results All adult onset groups exhibited decreased staining intensities to mAQP4 expressing cells. All groups reacted extensively with rAQP4 expressing cells. The patients with longer duration in group ON exhibited greater staining intensity with rAQP4 expressing cells, and the reverse phenomenon was observed in group M. Conclusions AQP4-Abs from each adult-onset group were considered to produce the antibodies recognizing different epitopes at least at the initial stage, and that could be a determinant of the various clinical subtypes of AQP4-related NMO spectrum disorders (NMOSDs). [ABSTRACT FROM AUTHOR]

Cysteinyl leukotrienes (CysLTs) are potent mediators of inflammation synthesized by the concerted actions of 5-lipoxygenase (5-LO), 5-LO-activating protein (FLAP), leukotriene C4 synthase, and additional downstream enzymes, starting with arachidonic acid substrate. CysLTs produced by macrophages, eosinophils, mast cells, and other inflammatory cells activate 3 different high-affinity CysLT receptors: CysLT1R, CysLT2R, and GPR 17. We sought to investigate vascular sites of CysLT2R expression and the role and mechanism of this receptor in mediating vascular permeability events. Vascular expression of CysLT2R was investigated by reporter gene expression in a novel CysLT2R deficient-LacZ mouse model. CysLT2R was expressed in small, but not large, vessels in mouse brain, bladder, skin, and cremaster muscle. Intravital, in addition to confocal and electron, microscopy investigations using FITC-labeled albumin in cremaster postcapillary venule preparations indicated rapid CysLT-mediated permeability, which was blocked by application of BAY-u9773, a dual CysLT1R/CysLT2R antagonist or by CysLT2R deficiency. Endothelial human CysLT2R over-expression in mice exacerbated vascular leakage even in the absence of exogenous ligand. The enhanced vascular permeability mediated by CysLT2R takes place via a transendothelial vesicle transport mechanism as opposed to a paracellular route and is controlled via Ca2+ signaling. Our results reveal that CysLT2R can mediate inflammatory reactions in a vascular bed-specific manner by altering transendothelial vesicle transport-based vascular permeability. [ABSTRACT FROM AUTHOR]

Little is known about the cerebral distribution and clearance of guanidinoacetate (GAA), the accumulation of which induces convulsions. The purpose of the present study was to identify creatine transporter (CRT)-mediated GAA transport and to clarify its cerebral expression and role in GAA efflux transport at the blood-cerebrospinal fluid barrier (BCSFB). CRT mediated GAA transport with a Km value of 269 μM/412 μM which was approximately 10-fold greater than that of CRT for creatine. There was wide and distinct cerebral expression of CRT and localization of CRT on the brush-border membrane of choroid plexus epithelial cells. The in vivo elimination clearance of GAA from the CSF was 13-fold greater than that ofd-mannitol reflecting bulk flow of the CSF. This process was partially inhibited by creatine. The characteristics of GAA uptake by isolated choroid plexus and an immortalized rat choroid plexus epithelial cell line (TR-CSFB cells) used as an in vitro model of BCSFB are partially consistent with those of CRT. These results suggest that CRT plays a role in the cerebral distribution of GAA and GAA uptake by the choroid plexus. However, in the presence of endogenous creatine in the CSF, CRT may make only a limited contribution to the GAA efflux transport at the BCSFB. [ABSTRACT FROM AUTHOR]

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that is widely expressed in the brain, and plays key roles in various cellular processes in response to both extracellular and intracellular stimuli. Here, we explored the role of FAK in cerebellar development. In the mouse cerebellum, FAK was found to be distributed as tiny cytoplasmic aggregates in various neuronal and glial elements, including Purkinje cells (PCs), Bergmann glia (BG), parallel fiber (PF)-terminals and climbing fiber (CF)-terminals. The neuron/glia-specific ablation of FAK impaired cerebellar foliation, such as variable decreases in foliation sizes and the lack of intercrural and precentral fissures. Some of the BG cells became situated ectopically in the molecular layer. Furthermore, the FAK ablation altered the innervation territories of CFs and PFs on PCs. CF innervation regressed to the basal portion of proximal dendrites and somata, whereas ectopic spines protruded from proximal dendrites and PFs expanded their territory by innervating the ectopic spines. Furthermore, the persistence of surplus CFs innervating PC somata caused multiple innervation. When FAK was selectively ablated in PCs, diminished dendritic innervation and persistent somatic innervation by CFs were observed, whereas cerebellar foliation and cell positioning of BG were normally retained. These results suggest that FAK in various neuronal and glial elements is required for the formation of normal histoarchitecture and cytoarchitecture in the cerebellum, and for the construction of proper innervation territory and synaptic wiring in PCs. [ABSTRACT FROM AUTHOR]

Brain-derived neurotrophic factor (BDNF) is involved in synaptic development and plasticity, and alterations in BDNF expression or signaling are implicated in drug addiction and psychiatric diseases, such as depression and schizophrenia. In this study, we administered phencyclidine to postnatal and adult rats with different time schedules, and determined the correlations between BDNF expression and the behavioral effects. Both single and repeated phencyclidine injections into adult rats induced BDNF up-regulation in the corticolimbic system and a decrease in prepulse inhibition, both of which were transient. In contrast, subchronic postnatal administration increased BDNF protein and mRNA levels in the hippocampus and entorhinal cortex, which were sustained until 8 weeks of age. In parallel, the postnatal rats treated with phencyclidine developed a persistent decrease in prepulse inhibition at the adult stage. The chronic BDNF increase appeared to contribute to the prepulse inhibition abnormality, as subchronic BDNF infusion into the hippocampus of normal rats mimicked the prepulse inhibition deficits. This study suggests that phencyclidine exposure during brain development induces sustained BDNF up-regulation in the limbic system with a biological link to sensorimotor gating deficits. [ABSTRACT FROM AUTHOR]

Transmembrane α-amino-3-hydroxyl-5-isoxazolepropionate (AMPA) receptor regulatory proteins (TARPs) play pivotal roles in AMPA receptor trafficking and gating. Here we examined cellular and subcellular distribution of TARP γ-8 in the mouse brain. Immunoblot and immunofluorescence revealed the highest concentration of γ-8 in the hippocampus. Immunogold electron microscopy demonstrated dense distribution of γ-8 on the synaptic and extrasynaptic surface of hippocampal neurons with very low intracellular labeling. Of the neuronal surface, γ-8 was distributed at the highest level on asymmetrical synapses of pyramidal cells and interneurons, whereas their symmetrical synapses selectively lacked immunogold labeling. Then, the role of γ-8 in AMPA receptor expression was pursued in the hippocampus using mutant mice defective in the γ-8 gene. In the mutant cornu ammonis (CA)1 region, synaptic and extrasynaptic AMPA receptors on dendrites and spines were severely reduced to 35–37% of control levels, whereas reduction was mild for extrasynaptic receptors on somata (74%) and no significant decrease was seen for intracellular receptors within spines. In the mutant CA3 region, synaptic AMPA receptors were reduced mildly at asymmetrical synapses in the stratum radiatum (67% of control level), and showed no significant decrease at mossy fiber–CA3 synapses. Therefore, γ-8 is abundantly distributed on hippocampal excitatory synapses and extrasynaptic membranes, and plays an important role in increasing the number of synaptic and extrasynaptic AMPA receptors on dendrites and spines, particularly, in the CA1 region. Variable degrees of reduction further suggest that other TARPs may also mediate this function at different potencies depending on hippocampal subregions, input sources and neuronal compartments. [ABSTRACT FROM AUTHOR]

Abstract We examined the regulation of the acoustic startle response in mutant mice of the N-methyl-d-aspartate (NMDA)- and δ-subtypes of the glutamate receptor (GluR) channel, which play important roles in neural plasticity in the forebrain and the cerebellum, respectively. Heterozygous mutant mice with reduced GluRε2 subunits of the NMDA receptor showed strongly enhanced startle responses to acoustic stimuli. On the other hand, heterozygous and homozygous mutation of the other NMDA receptor GluRε subunits exerted no, or only small effects on acoustic startle responses. The threshold of the auditory brainstem response of the GluRε2-mutant mice was comparable to that of the wild-type littermates. The primary circuit of the acoustic startle response is a relatively simple oligosynaptic pathway located in the lower brainstem, whilst the expression of GluRε2 is restricted to the forebrain. We thus suggest that the NMDA receptor GluRε2 subunit plays a role in the regulation of the startle reflex. Ablation of the cerebellar Purkinje cell-specific δ2 subunit of the GluR channel exerted little effect on the acoustic startle response but resulted in the enhancement of prepulse inhibition of the reflex. Because inhibition of the acoustic startle response by a weak prepulse is a measure of sensorimotor gating, the process by which an organism filters sensory information, these observations indicate the involvement of the cerebellum in the modulation of sensorimotor gating. [ABSTRACT FROM AUTHOR]

Cerebellar N-methyl-D-aspartate (NMDA) receptors are concentrated in the granular layer and are involved in motor coordination and the induction of long-term potentiation at mossy fibre-granule cell synapses. In the present study, we used immunohistochemistry to examine the distribution of NMDA receptor subunits in the adult mouse cerebellum. We found that appropriate pepsin pretreatment of sections greatly enhanced the sensitivity and specificity of immunohistochemical detection. As a result, intense immunolabelling for GluRs1 (NR2A), GluR epsilon 3 (NR2C), and GluR zeta 1 (NR1) all appeared in synaptic glomeruli of the granular layer. Double immunofluorescence showed that these subunits were colocalized in individual synaptic glomeruli. Within the glomerulus, NMDA receptor subunits were located between centrally- located huge mossy fibre terminals and peripherally-located tiny Golgi axon terminals. By immunoelectron microscopy, all three subunits were detected at the postsynaptic junction in granule cell dendrites, forming synapses with mossy fibre terminals. Consistent with the known functional localization, GluR epsilon 1, GluR epsilon 3, and GluR zeta 1 are, thus, anatomically concentrated at the mossy fibre-granule cell synapse. By contrast, immunohistochemical signals were very low in Purkinje cell somata and dendrites in the molecular layer. The lack of GluR zeta 1 immunolabelling in Purkinje cells was unexpected because the cells express GluR zeta 1 mRNA at high levels and high levels of GluR zeta 1 protein in the molecular layer were revealed by immunoblot. As Purkinje cells are exceptionally lacking GluRs expression, the discrepant result may provide in vivo evidence suggesting the importance of accompanying GluR epsilon subunits in synaptic localization of GluR zeta 1. [ABSTRACT FROM AUTHOR]

Abstract Following cell surface receptor activation, the α subunit of the Gq subclass of GTP-binding proteins activates the phosphoinositide signalling pathway. Here we examined the expression and localization of Gq protein α subunits in the adult mouse brain by in situ hybridization and immunohistochemistry. Of the four members of the Gq protein α subunits, Gαq and Gα11 were transcribed predominantly in the brain. The highest transcriptional level of Gαq was observed in cerebellar Purkinje cells (PCs) and hippocampal pyramidal cells, while that of Gα11 was noted in hippocampal pyramidal cells. Antibody against the C-terminal peptide common to Gαq and Gα11 strongly labelled the cerebellar molecular layer and hippocampal neuropil layers. In these regions, immunogold preferentially labelled the cytoplasmic face of postsynaptic cell membrane of PCs and pyramidal cells. Immunoparticles were distributed along the extra-junctional cell membrane of spines, dendrites and somata, but were almost excluded from the junctional membrane. By double immunofluorescence, Gαq/Gα11 was extensively colocalized with metabotropic glutamate receptor mGluR1α in dendritic spines of PCs and with mGluR5 in those of hippocampal pyramidal cells. Together with concentrated localization of mGluR1α and mGluR5 in a peri-junctional annulus on PC and pyramidal cell synapses (Baude et al. 1993, Neuron, 11, 771–787; Luján et al. 1996, Eur. J. Neurosci., 8, 1488–1500), the present molecular-anatomical findings suggest that peri-junctional stimulation of the group I metabotropic glutamate receptors is mediated by Gαq and/or Gα11, leading to the activation of the intracellular effector, phospholipase Cβ. [ABSTRACT FROM AUTHOR]

Abstract Nociceptive primary afferents have the capacity to induce a state of increased excitability in dorsal horn neurons of the spinal cord or central sensitization causing thermal hyperalgesia and touch-evoked pain (allodynia). It is believed that primary afferent C-fibres become hypersensitive and induce hyperalgesia and that low-threshold Aβ-fibres are responsible for induction of allodynia, the mechanisms of which remain elusive. We previously showed that intrathecal administration of prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α) induce allodynia in conscious mice. Here we demonstrated that selective elimination of C-fibres by neonatal capsaicin treatment resulted in the disappearance of allodynia induced by PGE2, but not that by PGF2α. PGE2-induced allodynia was not observed in N-methyl-D-aspartate (NMDA) receptor ε1 subunit knockout mice and was sensitive to morphine. In contrast, PGF2α-induced allodynia was not observed in NMDA ε4 subunit knockout mice and was insensitive to morphine. Furthermore, while PGF2α showed a capsaicin-insensitive feeble facilitatory action on evoked excitatory postsynaptic currents in dorsal horn neurons, PGE2 induced a long-lasting facilitation of evoked excitatory postsynaptic currents in a capsaicin-sensitive manner. Taken together, the present study demonstrates that there are two pathways for induction of allodynia and that capsaicin-sensitive C-fibres may participate in PGE2-induced allodynia. [ABSTRACT FROM AUTHOR]

Hippocampal synapses express two distinct forms of the long-term potentiation (LTP), i.e. NMDA receptor-dependent and -independent LTPs. To understand its molecular-anatomical basis, we produced affinity-purified antibodies against the GluRε1 (NR2A), GluRε2 (NR2B), and GluRζ1 (NR1) subunits of the N-methyl-d-aspartate (NMDA) receptor channel, and determined their distributions in the mouse hippocampus. Using NMDA receptor subunit-deficient mice as the specificity controls, section pretreatment with proteases (pepsin and proteinase K) was found to be very effective to detect authentic NMDA receptor subunits. As the result of modified immunohistochemistry, all three subunits were detected at the highest level in the strata oriens and radiatum of the CA1 subfield, and high levels were also seen in most other neuropil layers of the CA1 and CA3 subfields and of the dentate gyrus. However, the stratum lucidum, a mossy fibre-recipient layer of the CA3 subfield, contained low levels of the GluRε1 and GluRζ1 subunits and almost excluded the GluRε2 subunit. Double immunofluorescence with the AMPA receptor GluRα1 (GluR1 or GluR-A) subunit further demonstrated that the GluRε1 subunit was colocalized in a subset, not all, of GluRα1-immunopositive structures in the stratum lucidum. Therefore, the selective scarcity of these NMDA receptor subunits in the stratum lucidum suggests that a different synaptic targeting mechanism exerts within a single CA3 pyramidal neurone in vivo, which would explain contrasting significance of the NMDA receptor channel in LTP induction mechanisms between the mossy fibre-CA3 synapse and other hippocampal synapses. [ABSTRACT FROM AUTHOR]

In drug addiction, environmental stimuli previously associated with cocaine use readily elicit cocaine-associated memories, which persist long after abstinence and trigger cocaine craving and consumption. Although previous studies suggest that the medial prefrontal cortex (mPFC) is involved in the expression of cocaine-addictive behaviors, it remains unclear whether excitatory and inhibitory neurons in the mPFC are causally related to the formation and retrieval of cocaine-associated memories. To address this issue, we used the designer receptors exclusively activated by designer drugs (DREADD) technology combined with a cocaine-induced conditioned place preference (CPP) paradigm. We suppressed mPFC neuronal activity in a cell-type- and timing-dependent manner. C57BL/6J wild-type mice received bilateral intra-mPFC infusion of an adeno-associated virus (AAV) expressing inhibitory DREADD (hM4Di) under the control of CaMKII promotor to selectively suppress mPFC pyramidal neurons. GAD67-Cre mice received bilateral intra-mPFC infusion of a Cre-dependent AAV expressing hM4Di to specifically silence GABAergic neurons. Chemogenetic suppression of mPFC pyramidal neurons significantly attenuated both the acquisition and expression of cocaine CPP, while suppression of mPFC GABAergic neurons affected neither the acquisition nor expression of cocaine CPP. Moreover, chemogenetic inhibition of mPFC glutamatergic neurons did not affect the acquisition and expression of lithium chloride-induced conditioned place aversion. These results suggest that the activation of glutamatergic, but not GABAergic, neurons in the mPFC mediates both the formation and retrieval of cocaine-associated memories. [ABSTRACT FROM AUTHOR]