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Cognitive dysfunction is a core feature of dementia and a prominent feature in psychiatric disease. As non-redundant regulators of intracellular cAMP gradients, phosphodiesterases (PDE) mediate fundamental aspects of brain function relevant to learning, memory, and higher cognitive functions. Phosphodiesterase-4B (PDE4B) is an important phosphodiesterase in the hippocampal formation, is a major Disrupted in Schizophrenia 1 (DISC1) binding partner and is itself a risk gene for psychiatric illness. To define the effects of specific inhibition of the PDE4B subtype, we generated mice with a catalytic domain mutant form of PDE4B (Y358C) that has decreased ability to hydrolyze cAMP. Structural modeling predictions of decreased function and impaired binding with DISC1 were confirmed in cell assays. Phenotypic characterization of the PDE4BY358C mice revealed facilitated phosphorylation of CREB, decreased binding to DISC1, and upregulation of DISC1 and β-Arrestin in hippocampus and amygdala. In behavioral assays, PDE4BY358C mice displayed decreased anxiety and increased exploration, as well as cognitive enhancement across several tests of learning and memory, consistent with synaptic changes including enhanced long-term potentiation and impaired depotentiation ex vivo. PDE4BY358C mice also demonstrated enhanced neurogenesis. Contextual fear memory, though intact at 24 h, was decreased at 7 days in PDE4BY358C mice, an effect replicated pharmacologically with a non-selective PDE4 inhibitor, implicating cAMP signaling by PDE4B in a very late phase of consolidation. No effect of the PDE4BY358C mutation was observed in the prepulse inhibition and forced swim tests. Our data establish specific inhibition of PDE4B as a promising therapeutic approach for disorders of cognition and anxiety, and a putative target for pathological fear memory.

Missense mutations in ATP1A3 encoding Na+,K+-ATPase α3 are the primary cause of alternating hemiplegia of childhood (AHC). Most ATP1A3 mutations in AHC lie within a cluster in or near transmembrane α-helix TM6, including I810N that is also found in the Myshkin mouse model of AHC. These mutations all substantially reduce Na+,K+-ATPase α3 activity. Herein, we show that Myshkin mice carrying a wild-type Atp1a3 transgene that confers a 16 % increase in brain-specific total Na+,K+-ATPase activity show significant phenotypic improvements compared with non-transgenic Myshkin mice. Interventions to increase the activity of wild-type Na+,K+-ATPase α3 in AHC patients should be investigated further. Electronic supplementary material The online version of this article (doi:10.1007/s10048-015-0461-1) contains supplementary material, which is available to authorized users.

The goal of the Complex Trait Consortium is to promote the development of resources that can be used to understand, treat and ultimately prevent pervasive human diseases. Existing and proposed mouse resources that are optimized to study the actions of isolated genetic loci on a fixed background are less effective for studying intact potygenic networks and interactions among genes, environments, pathogens and other factors. The Collaborative Cross will provide a common reference panel specifically designed for the integrative analysis of complex systems and will change the way we approach human health and disease.

This study investigated the function of the adhesion molecule L1 in unmyelinated fibers of the peripheral nervous system (PNS) by analysis of L1- deficient mice. We demonstrate that L1 is present on axons and Schwann cells of sensory unmyelinated fibers, but only on Schwann cells of sympathetic unmyelinated fibers. In L1-deficient sensory nerves, Schwann cells formed but failed to retain normal axonal ensheathment. L1-deficient mice had reduced sensory function and loss of unmyelinated axons, while sympathetic unmyelinated axons appeared normal. In nerve transplant studies, loss of axonal-L1, but not Schwann cell-L1, reproduced the L1-deficient phenotype. These data establish that heterophilic axonal-L1 interactions mediate adhesion between unmyelinated sensory axons and Schwann cells, stabilize the polarization of Schwann cell surface membranes, and mediate a trophic effect that assures axonal survival.

Cognitive deficit is a core of schizophrenia and it is not effectively treated by the available antipsychotic drugs, hence new and more effective therapy is needed. Schizophrenia is considered as a pathway disorder where Disrupted-In-Schizophrenia-1 (DISC1) is important molecular player that regulates multiple cellular cascades. We recently reported synergistic action between phosphodiesterase-4 (PDE4) and glycogen synthase kinase-3 (GSK-3) as DISC1 interacting proteins. In the current study we characterized behavioural effects of a newly developed compound, VP1.15 that inhibits both PDE7 and GSK-3 with main focus on its antipsychotic and cognitive capacities. VP1.15 reduced ambulation in C57BL/6J mice in a dose-dependent manner (7.5 mg/kg and 3 mg/kg, respectively) and, hence, lower dose was chosen for the further analysis. VP1.1.5 facilitated pre-pulse inhibition (PPI), reversed amphetamine- but not MK-801-induced PPI deficit. The drug was able to ameliorate the disrupted latent inhibition (LI) induced by the increased number of conditioning trials and reversed amphetamine-induced LI deficit, supporting further its antipsychotic effects. The drug also significantly improved episodic memory in the spatial object recognition test, facilitated working memory in Y-maze and enhanced cued fear memory, but had no effect on executive function in the Puzzle box and contextual fear conditioning. Taken together, VP1.15 elicited antipsychotic effects and also facilitated cognitive domains in mice, suggesting that multitarget drugs, affecting molecular substrates from the same pathway, perhaps could be antipsychotics of new-generation that open a new possibilities in drug discoveries. This article is part of a Special Issue entitled 'Cognitive Enhancers'. © 2012 Elsevier Ltd. All rights reserved.

Disrupted-in-schizophrenia-1 (DISC1) is associated with mental disorders, including major depression. We previously showed that DISC1-Q31L mutant mice have depression-like behaviors and can therefore be used to study neurobiological mechanisms of depression and antidepressant (AD) medication action. First, we found reduced levels of dopamine, serotonin and norepinephrine in the nucleus accumbens (NAC) of DISC1-Q31L mutants. Next, we assessed social-conditioned place preference as a reward-dependent task and the capacity of distinct ADs to correct impaired social behavior in DISC1-Q31L mice. Bupropion, but not fluoxetine or desipramine, was able to correct deficient social facilitation, social reward, and social novelty in DISC1-Q31L mutants, whereas all three ADs were able to improve social motivation and behavioral despair in DISC1-Q31L mutants. Furthermore, we sought to correlate social anhedonia with molecular and cellular features including dendritic spine density, β-arrestin-1,2, and cAMP-response-element-binding protein (CREB) in the NAC as biomarkers related to depression and the DISC1 pathway. DISC1-Q31L mutants showed reduced levels of β-arrestin-1,2, CREB, and spine density in the NAC, further supporting the construct validity of the genetic model. Bupropion induced the greatest effect on CREB in DISC1-Q31L mutants, whereas all studied ADs corrected the reduced levels of β-arrestin-1,2 and modestly ameliorated deficient spine density in this brain region. Overall, we find neurobiological changes accompanying social anhedonia in the NAC of DISC1-Q31L mutant mice, consistent with a role for DISC1 in regulating social reward as an endophenotype of depression.

Myshkin mice heterozygous for an inactivating mutation in the neuron-specific Na(+) ,K(+) -ATPase α3 isoform show behavior analogous to mania, including an abnormal endogenous circadian period. Agrin is a proteoglycan implicated as a regulator of synapses that has been proposed to inhibit activity of Na(+) ,K(+) -ATPase α3. We examined whether the mania-related behavior of Myshkin mice could be rescued by a reduction in the expression of agrin through genetic knockout. The suppression of agrin reduced hyperambulation and holeboard exploration, restored anxiety-like behavior (or reduced risk-taking behavior), improved prepulse inhibition and shortened the circadian period. Hence, agrin is important for regulating mania-like behavior and circadian rhythms. In Myshkin mice, the suppression of agrin increased brain Na(+) ,K(+) -ATPase activity by 11 ± 4%, whereas no effect on Na(+) ,K(+) -ATPase activity was detected when agrin was suppressed in mice without the Myshkin mutation. These results introduce agrin as a potential therapeutic target for the treatment of mania and other neurological disorders associated with reduced Na(+) ,K(+) -ATPase activity and neuronal hyperexcitability.

Recent studies have identified disrupted-in-schizophrenia-1 (DISC1) as a strong genetic risk factor associated with schizophrenia. Previously, we have reported that a mutation in the second exon of the DISC1 gene [leucine to proline at amino acid position 100, L100P] leads to the development of schizophrenia-related behaviors in mice. Glycogen synthase kinase-3 (GSK-3) is a serine/threonine protein kinase that interacts with the N-terminal region of DISC1 (aa 1-220) and has been implicated as an important downstream component in the etiology of schizophrenia. Here, for the first time, we show that pharmacological and genetic inactivation of GSK-3 reverse prepulse inhibition and latent inhibition deficits as well as normalizing the hyperactivity of Disc1-L100P mutants. In parallel to these observations, interaction between DISC1 and GSK-3α and β is reduced in Disc1-L100P mutants. Our data provide genetic, biochemical, and behavioral evidence for a molecular link between DISC1 and GSK-3 in relation to psychopathology and highlights the value of missense mutations in dissecting the underlying and complex molecular mechanisms of neurological disorders.

International audience; Schwann cells elaborate myelin sheaths around axons by spirally wrapping and compacting their plasma membranes. Although actin remodeling plays a crucial role in this process, the effectors that modulate the Schwann cell cytoskeleton are poorly defined. Here, we show that the actin cytoskeletal regulator, neural Wiskott-Aldrich syndrome protein (N-WASp), is upregulated in myelinating Schwann cells coincident with myelin elaboration. When N-WASp is conditionally deleted in Schwann cells at the onset of myelination, the cells continue to ensheath axons but fail to extend processes circumferentially to elaborate myelin. Myelin-related gene expression is also severely reduced in the N-WASp-deficient cells and in vitro process and lamellipodia formation are disrupted. Although affected mice demonstrate obvious motor deficits these do not appear to progress, the mutant animals achieving normal body weights and living to advanced age. Our observations demonstrate that N-WASp plays an essential role in Schwann cell maturation and myelin formation.

As the genomic basis for Down syndrome (DS), human trisomy 21 is the most common genetic cause of intellectual disability in children and young people. The genomic regions on human chromosome 21 (Hsa21) are syntenic to three regions in the mouse genome, located on mouse chromosome 10 (Mmu10), Mmu16, and Mmu17. Recently, we have developed three new mouse models using chromosome engineering carrying the genotypes of Dp(10)1Yey/+, Dp(16)1Yey/+, or Dp(17)1Yey/+, which harbor a duplication spanning the entire Hsa21 syntenic region on Mmu10, Mmu16, or Mmu17, respectively. In this study, we analyzed the hippocampal long-term potentiation (LTP) and cognitive behaviors of these models. Our results show that, while the genotype of Dp(17)1Yey/+ results in abnormal hippocampal LTP, the genotype of Dp(16)1Yey/+ leads to both abnormal hippocampal LTP and impaired learning/memory. Therefore, these mutant mice can serve as powerful tools for further understanding the mechanism underlying cognitively relevant phenotypes associated with DS, particularly the impacts of different syntenic regions on these phenotypes.

Copy-number variation in the human genome can be disease-causing or phenotypically neutral. This type of genetic rearrangement associated with human chromosome 21 (Hsa21) underlies partial Monosomy 21 and Trisomy 21. Mental retardation is a major clinical manifestation of partial Monosomy 21. To model this human chromosomal deletion disorder, we have generated novel mouse mutants carrying heterozygous deletions of the 2.3- and 1.1-Mb segments on mouse chromosome 10 (Mmu10) and Mmu17, respectively, which are orthologous to the regions on human 21q22.3, using Cre/loxP-mediated chromosome engineering. Alterations of the transcriptional levels of genes within the deleted intervals reflect gene-dosage effects in the mutant mice. The analysis of cognitive behaviors shows that the mutant mice carrying the deletion on either Mmu10 or Mmu17 are impaired in learning and memory. Therefore, these mutants represent mouse models for Monosomy 21-associated mental retardation, which can serve as a powerful tool to study the molecular mechanism underlying the clinical phenotype and should facilitate efforts to identify the haploinsufficient causative genes.

Background Glycogen synthase kinase-3 (GSK-3) is a widely expressed and highly conserved serine/threonine protein kinase encoded by two genes that generate two related proteins: GSK-3α and GSK-3β. Mice lacking a functional GSK-3α gene were engineered in our laboratory; they are viable and display insulin sensitivity. In this study, we have characterized brain functions of GSK-3α KO mice by using a well-established battery of behavioral tests together with neurochemical and neuroanatomical analysis. Results Similar to the previously described behaviours of GSK-3β+/-mice, GSK-3α mutants display decreased exploratory activity, decreased immobility time and reduced aggressive behavior. However, genetic inactivation of the GSK-3α gene was associated with: decreased locomotion and impaired motor coordination, increased grooming activity, loss of social motivation and novelty; enhanced sensorimotor gating and impaired associated memory and coordination. GSK-3α KO mice exhibited a deficit in fear conditioning, however memory formation as assessed by a passive avoidance test was normal, suggesting that the animals are sensitized for active avoidance of a highly aversive stimulus in the fear-conditioning paradigm. Changes in cerebellar structure and function were observed in mutant mice along with a significant decrease of the number and size of Purkinje cells. Conclusion Taken together, these data support a role for the GSK-3α gene in CNS functioning and possible involvement in the development of psychiatric disorders.

Reduced function of the N-methyl-d-aspartate receptor (NMDAR) has been implicated in the pathophysiology of schizophrenia. The NMDAR contains a glycine binding site in its NR1 subunit that may be a useful target for the treatment of schizophrenia. In this study, we assessed the therapeutic potential of long-term increases in the brain levels of the endogenous NMDAR glycine site agonist D-serine, through the genetic inactivation of its catabolic enzyme D-amino acid oxidase (DAO) in mice. The effects of eliminating DAO function were investigated in mice that display schizophrenia-related behavioral deficits due to a mutation (Grin 1(D481N)) in the NR1 subunit that results in a reduction in NMDAR glycine affinity. Grin 1(D481N) mice show deficits in sociability, prolonged latent inhibition, enhanced startle reactivity and impaired spatial memory. The hypofunctional Dao 1(G181R) mutation elevated brain levels of D-serine, but alone it did not affect performance in the behavioral measures. Compared to animals with only the Grin 1(D481N) mutation, mice with both the Dao1(G181R) and Grin 1(D481N) mutations displayed an improvement in social approach and spatial memory retention, as well as a reversal of abnormally persistent latent inhibition and a partial normalization of startle responses. Thus, an increased level of D-serine resulting from decreased catalysis corrected the performance of mice with deficient NMDAR glycine site activation in behavioral tasks relevant to the negative and cognitive symptoms of schizophrenia. Diminished DAO activity and elevations in D-serine may serve as an effective therapeutic intervention for the treatment of psychiatric symptoms.

Abnormal N-methyl-d-aspartate receptor (NMDAR) function has been implicated in the pathophysiology of schizophrenia. d-serine is an important NMDAR modulator, and to elucidate the role of the d-serine synthesis enzyme serine racemase (Srr) in schizophrenia, we identified and characterized mice with an ENU-induced mutation that results in a complete loss of Srr activity and dramatically reduced d-serine levels. Mutant mice displayed behaviors relevant to schizophrenia, including impairments in prepulse inhibition, sociability and spatial discrimination. Behavioral deficits were exacerbated by an NMDAR antagonist and ameliorated by d-serine or the atypical antipsychotic clozapine. Expression profiling revealed that the Srr mutation influenced several genes that have been linked to schizophrenia and cognitive ability. Transcript levels altered by the Srr mutation were also normalized by d-serine or clozapine treatment. Furthermore, analysis of SRR genetic variants in humans identified a robust association with schizophrenia. This study demonstrates that aberrant Srr function and diminished d-serine may contribute to schizophrenia pathogenesis.

In an attempt to identify a molecule in target recognition by CD3- large granular lymphocytes (LGL), we have generated a rabbit antiidiotypic (anti-ID) serum against a monoclonal antibody (mAb 36) that reacted with the cell membrane of K562. Flow cytometry analysis demonstrated that the anti-ID serum bound selectively to CD3- LGL and that F(ab')2 fragments of the anti-ID serum blocked both target cell binding and lysis by NK cells. Stimulation of CD3- LGL with F(ab')2 fragments resulted in the release of serine esterases and the secretion of interferon gamma. Furthermore, anti-ID F(ab')2 antibodies crosslinked to anti-DNP F(ab')2 mediated directed cytotoxicity of a non-natural killer (NK)-susceptible mouse target (YAC-1) via this surface ligand. These functional reactivities were only removed by adsorption with the specific idiotype. Protein analysis showed that the anti-ID serum immunoprecipitated 80-, 110-, and 150-kD proteins. Using this anti-ID, a partial cDNA was cloned and an antipeptide antiserum was made against the portion of the predicted amino acid sequence that corresponded to a portion of the ID binding region. This antipeptide serum exhibited similar functional and biochemical reactivities to those observed with the anti-ID serum. These data suggest that the cell surface moiety recognized by the anti-ID and anti-p104 is novel and is selectively involved in both recognition and triggering of NK-mediated lytic function.

Huntingtin-interacting protein 1 (HIP1) is an endocytic adaptor protein that plays a role in clathrin-mediated endocytosis and the ligand-induced internalization of AMPA receptors (AMPARs) (Metzler et al., 2003). In the present study, we investigated the role of HIP1 in NMDA receptor (NMDAR) function by analyzing NMDA-dependent transport and NMDA-induced excitotoxicity in neurons from HIP1−/−mice. HIP1 colocalizes with NMDARs in hippocampal and cortical neurons and affinity purifies with NMDARs by GST (glutathioneS-transferase) pull down and coimmunoprecipitation. A profound decrease in NMDA-induced AMPAR internalization of 75% occurs in neurons from HIP1−/−mice compared with wild type, using a quantitative single-cell-based internalization assay. This defect in NMDA-dependent removal of surface AMPARs is in agreement with the observed defect in long-term depression induction in hippocampal brain slices of HIP1−/−mice and supports a role of HIP1 in AMPAR internalizationin vivo. HIP1−/−neurons are partially protected from NMDA-induced excitotoxicity as assessed by LDH (lactate dehydrogenase) release, TUNEL (terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick end labeling) and caspase-3 activation assays, which points to a role of HIP1 in NMDA-induced cell death. Interestingly, phosphorylation of Akt and its substrate huntingtin (htt) decreases during NMDA-induced excitotoxicity by 48 and 31%, respectively. This decrease is significantly modulated by HIP1, resulting in 94 and 48% changes in P-Akt and P-htt levels in HIP1−/−neurons, respectively. In summary, we have shown that HIP1 influences important NMDAR functions and that both HIP1 and htt participate in NMDA-induced cell death. These findings may provide novel insights into the cellular mechanisms underlying enhanced NMDA-induced excitotoxicity in Huntington's disease.

Understanding basic neuronal mechanisms hold the hope for future treatment of brain disease. The 1st international conference on synapse, memory, drug addiction and pain was held in beautiful downtown Toronto, Canada on August 21-23, 2006. Unlike other traditional conferences, this new meeting focused on three major aims: (1) to promote new and cutting edge research in neuroscience; (2) to encourage international information exchange and scientific collaborations; and (3) to provide a platform for active scientists to discuss new findings. Up to 64 investigators presented their recent discoveries, from basic synaptic mechanisms to genes related to human brain disease. This meeting was in part sponsored by Molecular Pain, together with University of Toronto (Faculty of Medicine, Department of Physiology as well as Center for the Study of Pain). Our goal for this meeting is to promote future active scientific collaborations and improve human health through fundamental basic neuroscience researches. The second international meeting on Neurons and Brain Disease will be held in Toronto (August 29-31, 2007). © 2006 Bi et al; licensee BioMed Central Ltd.

At CA1 synapses, activation of NMDA receptors (NMDARs) is required for the induction of both long-term potentiation and depression. The basal level of activity of these receptors is controlled by converging cell signals from G-protein-coupled receptors and receptor tyrosine kinases. Pituitary adenylate cyclase activating peptide (PACAP) is implicated in the regulation of synaptic plasticity because it enhances NMDAR responses by stimulating Gαs-coupled receptors and protein kinase A (Yaka et al., 2003). However, the major hippocampal PACAP1 receptor (PAC1R) also signals via Gαqsubunits and protein kinase C (PKC). In CA1 neurons, we showed that PACAP38 (1 nm) enhanced synaptic NMDA, and evoked NMDAR, currents in isolated CA1 neurons via activation of the PAC1R, Gαq, and PKC. The signaling was blocked by intracellular applications of the Src inhibitory peptide Src(40-58). Immunoblots confirmed that PACAP38 biochemically activates Src. A Gαqpathway is responsible for this Src-dependent PACAP enhancement because it was attenuated in mice lacking expression of phospholipase C β1, it was blocked by preventing elevations in intracellular Ca2+, and it was eliminated by inhibiting either PKC or cell adhesion kinase β [CAKβ or Pyk2 (proline rich tyrosine kinase 2)]. Peptides that mimic the binding sites for either Fyn or Src on receptor for activated C kinase-1 (RACK1) also enhanced NMDAR in CA1 neurons, but their effects were blocked by Src(40-58), implying that Src is the ultimate regulator of NMDARs. RACK1 serves as a hub for PKC, Fyn, and Src and facilitates the regulation of basal NMDAR activity in CA1 hippocampal neurons.

Secretion in neutrophils is thought to be regulated in different ways for the different granule types. Specific granules are endowed with proteins which are related to docking and fusion events and are absent on azurophilic granules. Furthermore, even if secretion of content from all neutrophil granules is a Ca(2+)-dependent process, a higher concentration of cytosolic calcium is required for azurophilic than for specific granule secretion. In this paper we show that human neutrophils and promyelocitic cells express neuronal calcium sensor-1 (NCS-1), a calcium binding protein involved in exocytosis in various cell types. Both mRNA and protein were found in mature cells and precursors. NCS-1 is shown to be mainly associated with azurophilic granules and, therefore could play an instrumental role in the calcium-dependent secretion of azurophilic granules.

The role of rat neuronal calcium sensor-1 (NCS-1), a Ca2+-binding protein, in synapse formation and transmitter release was examined in mouse neuroblastoma × rat glioma hybrid NG108-15 cells in culture. Wild-type NG108-15 cells expressed rodent NCS-1. Endogenous NCS-1 was partially co-localized with the synaptic protein SNAP-25 at the plasma membrane in both cell bodies and processes, but not with the Golgi marker β-COP, an individual coat subunit of the coatomer complex present on Golgi-derived vesicles. In NG108-15 cells co-cultured with rat myotubes, partial co-localization of SNAP-25 and NCS-1 was observed at the plasma membrane of neurites and growth cones, some of which had synaptic contacts to muscle cells. Transient co-transfection of the rat NCS-1 cDNA and green fluorescent protein (GFP) resulted in NCS-1 overexpression in about 30 % of the cells as determined by fluorescence microscopy. The rate of functional synapse formation with co-cultured rat myotubes increased 2-fold as determined by the presence of miniature endplate potentials (MEPPs) in NCS-1-overexpressing NG108-15 cells compared to non- and mock-transfected cells. The number of neurites per cell, branches per neurite and length of neurites was slightly less in cells that were either transiently transfected (GFP-NCS-1-fluorescence positive) or stably transformed with NCS-1 compared to GFP-NCS-1-negative, non-transfected or mock-transfected NG108-15 cells. The number of action potentials that elicited endplate potentials increased in NG108-15 cells stably transformed with rat NCS-1. The mean number of quanta per impulse (m) increased 5-fold. These results show that NCS-1 functions to facilitate synapse formation, probably because of the increased quantal content of evoked acetylcholine release. Neuronal calcium sensor-1 (NCS-1) is a protein bearing four EF-hand binding motifs (McFerran et al. 1998) and functions as a calcium sensor, because it co-operatively binds two Ca2+ with affinity constants of 8.9 × 10−4 and 1.4 × 10−8m, respectively (Cox et al. 1994). NCS-1, originally termed frequenin, exhibited an enhanced frequency-dependent facilitation of neurotransmitter release, and was first described in Drosophila and Xenopus (Pongs et al. 1993; Angaut-Petit et al. 1993; Poulain et al. 1994; Olafssoet al. 1995) and more recently in Crustacea (Jeromin et al. 1999). The protein sequence of Drosophila frequenin contains 187 amino acids. NCS-1 (frequenin) is a member of a superfamily of related Ca2+-binding proteins. Within this family are two subclasses of proteins; type A includes proteins such as visinin, recoverin and s-modulin, which are expressed in photoreceptors; type B includes proteins expressed in neurons such as neurocalcin, hippocalcin, frequenin/NCS-1 and VILIP (Paterlini et al. 2000). Frequenin has been shown to be localized at the frog neuromuscular junction, muscle spindle and nerve (Werle et al. 2000) and to enhance synaptic efficacy at the Drosophila and Xenopus neuromuscular junction (Pongs et al. 1993; Rivosecchi et al. 1994; Angaut-Petit et al. 1998). Overexpression of frequenin in transgenic flies only increased evoked transmitter release, while acute infusion of Xenopus frequenin as a recombinant protein increased both spontaneous and evoked release. The basis for this discrepancy is unclear at present. Though frequenin was shown to co-localize with synaptophysin in vestibular efferent endings in mouse inner ear (Sage et al. 2000) and in rat brain and neuroendocrine (phaeochromocytoma PC12) cells (authors' unpublished results), it is not known whether it is related to synaptotagmin, a vesicle-associated protein that also binds Ca2+. In rat PC12 cells, overexpression of NCS-1 (Nef et al. 1995: Olafsson et al. 1997; McFerran et al. 1998, 1999; Martone et al. 1999) led to an increase in dense-core granule exocytosis (McFerran et al. 1998) only in intact, but not in permeabilized cells. The authors concluded that NCS-1/frequenin may play an indirect modulatory role in the secretion of dense-core vesicles. Whether or not NCS-1 plays a more direct role in neurotransmitter release, especially from clear vesicles containing small molecules such as acetylcholine or glutamate, has not been elucidated. To address this question, we have chosen the NG108-myocyte co-culture model (Nirenberg et al. 1983a, b) It has been reported that mouse neuroblastoma × rat glioma hybrid NG108-15 cells do not form functional autaptic synapses (Nelson et al. 1976). In contrast, co-culturing of NG108-15 cells with muscle cells leads to the formation of cholinergic synapses, involving intracellular cAMP-dependent signalling pathways (Nelson et al. 1976; Puro & Nirenberg, 1976; Higashida et al. 1981; Yano et al. 1984; Ogura et al. 1990; Kimura & Higashida, 1992). In the present study, we examined the role of NCS-1. First, we analysed the subcellular localization of endogenous NCS-1 in NG108-15 cells. Second, we established an overexpression method for rat NCS-1 by transient and stable transfection of the wild-type rat NCS-1 or rat NCS-1 co-expressed with green fluorescent protein (GFP) in NG108-15 cells. We demonstrate here an increase in functional synapse formation with myotubes and an increased acetylcholine release in transfected NG108-15 cells, by recording muscle endplate potentials arising from spontaneous or evoked release of acetylcholine.

Regulators of G protein signaling (RGS) proteins accelerate the GTPase activity of Gα protein subunitsin vitro, negatively regulating G protein-coupled receptor signaling. The physiological role of mammalian RGS proteins is largely unknown. The RGS family memberrgs2was cloned as an immediate early response gene up-regulated in T lymphocytes after activation. To investigate the role of RGS2in vivo, we generatedrgs2-deficient mice. We show that targeted mutation ofrgs2in mice leads to reduced T cell proliferation and IL-2 production, which translates in an impaired antiviral immunityin vivo. Interestingly,rgs2−/−mice also display increased anxiety responses and decreased male aggression in the absence of cognitive or motor deficits. RGS2 also controls synaptic development and basal electrical activity in hippocampal CA1 neurons. Thus, RGS2 plays an important role in T cell activation, synapse development in the hippocampus, and emotive behaviors.

Natural killer cells are non-major histocompatibility complex-restricted large granular lymphocytes that can recognize and destroy tumor cells without prior stimulation. A 150-kDa molecule on the surface of human natural killer cells was identified as a component of a putative tumor-recognition complex. We report here the isolation of cDNAs coding for the 150-kDa tumor-recognition molecule from human and mouse cDNA libraries. The amino terminus of the predicted protein contains a large hydrophobic region followed by a domain that is highly homologous to cyclophilin/peptidylprolyl cis-trans isomerase. The remainder of the protein is extremely hydrophilic and contains three homologous positively charged clusters. There are also three regions that contain extensive arginine- and serine-rich repeats. Comparison of the human and mouse predicted amino acid sequences revealed > 80% homology.

Mice with severe combined immunodeficiency syndrome (SCID) exhibit an impairment in both T and B cell maturation, whereas myelopoiesis remains unaffected. We report here that spleens from SCID mice have undergone phenotypic expansion of cells bearing the NK-2 and asialo GM1 markers (70-80%) characteristic of NK cells and this expansion is accompanied by a 3-4-fold enrichment in NK cytolytic activity over their normal C.B-17 littermates. Furthermore, the NK cells from SCID mice do not rearrange or express T cell receptor alpha or beta genes, or a third T cell rearranging gene, gamma. These findings suggest that (a) T cell receptors are not necessary for NK-mediated cytolysis, and (b) either NK cells constitute an entirely distinct lineage or NK cell function is acquired in pre-T cells prior to the expression of T cell receptor genes.

SummaryTo support the role of DISC1 in human psychiatric disorders, we identified and analyzed two independently derived ENU-induced mutations in Exon 2 of mouse Disc1. Mice with mutation Q31L showed depressive-like behavior with deficits in the forced swim test and other measures that were reversed by the antidepressant bupropion, but not by rolipram, a phosphodiesterase-4 (PDE4) inhibitor. In contrast, L100P mutant mice exhibited schizophrenic-like behavior, with profound deficits in prepulse inhibition and latent inhibition that were reversed by antipsychotic treatment. Both mutant DISC1 proteins exhibited reduced binding to the known DISC1 binding partner PDE4B. Q31L mutants had lower PDE4B activity, consistent with their resistance to rolipram, suggesting decreased PDE4 activity as a contributory factor in depression. This study demonstrates that Disc1 missense mutations in mice give rise to phenotypes related to depression and schizophrenia, thus supporting the role of DISC1 in major mental illness.

SummaryThe molecular underpinnings of exploration and its link to learning and memory remain poorly understood. Here we show that inducible, modest overexpression of neuronal calcium sensor 1 (Ncs1) selectively in the adult murine dentate gyrus (DG) promotes a specific form of exploratory behavior. The mice also display a selective facilitation of long-term potentiation (LTP) in the medial perforant path and a selective enhancement in rapid-acquisition spatial memory, phenotypes that are reversed by direct application of a cell-permeant peptide (DNIP) designed to interfere with NCS-1 binding to the dopamine type-2 receptor (D2R). Moreover, the DNIP and the D2R-selective antagonist L-741,626 attenuated exploratory behavior, DG LTP, and spatial memory in control mice. These data demonstrate a role for NCS-1 and D2R in DG plasticity and provide insight for understanding how the DG contributes to the origin of exploration and spatial memory acquisition.

AMPA receptors (AMPARs) are not thought to be involved in the induction of long-term potentiation (LTP), but may be involved in its expression via second messenger pathways. However, one subunit of the AMPARs, GluR2, is also known to control Ca2+ influx. To test whether GluR2 plays any role in the induction of LTP, we generated mice that lacked this subunit. In GluR2 mutants, LTP in the CA1 region of hippocampal slices was markedly enhanced (2-fold) and nonsaturating, whereas neuronal excitability and paired-pulse facilitation were normal. The 9-fold increase in Ca2+ permeability, in response to kainate application, suggests one possible mechanism for enhanced LTP. Mutant mice exhibited increased mortality, and those surviving showed reduced exploration and impaired motor coordination. These results suggest an important role for GluR2 in regulating synaptic plasticity and behavior.

A 6-thioguanine-resistant, human lymphoblastoid B-cell line (GM1500 6TG A-11; IgG secreting) was mutagen-treated with low-level gamma-irradiation and selected for ouabain resistance. One line showing 10,000-fold higher drug resistance, designated KR-4, was fused with an Epstein-Barr virus-transformed, cloned, B-lymphocyte cell line (B6) producing antitetanus toxoid (TT) antibody (IgM), and the hybrids were selected in hypoxanthine/aminopterin/thymidine medium containing 10 microM ouabain. Surviving cells, which arose at an optimal frequency of 10(-5), were subcloned by limiting dilution and screened for anti-TT production. Out of 395 final subclones, 372 were found positive for anti-TT, and seven that were selected for further study secreted specific antibody (IgM, kappa chain) at a maximum concentration of 3-6 micrograms/ml. The differential rate of anti-TT production during the logarithmic phase of cell growth was 15-fold higher in the hybridomas than in the original B6 line. The hybrid nature of the clones was confirmed by karyotype analysis, histocompatibility antigen typing, and expression of secreted and membrane-bound Ig classes. Biosynthetic labeling of the cells revealed that all hybrids secreted both IgM and IgG but that only the IgM class had specificity for TT. Because Epstein-Barr virus is a polyclonal B-lymphocyte activator, the technique we applied here may be useful for increasing the recovery of rare antigen-specific B cells in the peripheral blood and for improving the frequency and stability of hybridomas secreting a given antibody.

Background Interneuron deficits are one of the most consistent findings in post-mortem studies of schizophrenia patients and are likely important in the cognitive deficits associated with schizophrenia. Disrupted-in-Schizophrenia 1 (DISC1), a strong susceptibility gene for schizophrenia and other mental illnesses, is involved in neurodevelopment, including that of interneurons. However, the mechanism by which DISC1 regulates interneuron development remains unknown. In this study, we analyzed interneuron histology in the Disc1-L100P single point mutation mouse, that was previously shown to have behavioral abnormalities and cortical developmental defects related to schizophrenia. Results We sought to determine whether a Disc1-L100P point mutation in the mouse would alter interneuron density and location. First, we examined interneuron position in the developing mouse cortex during embryonic days 14–16 as an indicator of interneuron tangential migration, and found striking migration deficits in Disc1-L100P mutants. Further analysis of adult brains revealed that the Disc1-L100P mutants have selective alterations of calbindin- and parvalbumin-expressing interneurons in the cortex and hippocampus, decreased GAD67/PV co-localization and mis-positioned interneurons across the neocortex when compared to wild-type littermates. Conclusion Our results are consistent with the anomalies seen in post-mortem schizophrenia studies and other Disc1 mutant mouse models. Future research is required to determine the specific mechanisms underlying these cellular deficits. Overall, these findings provide further evidence that DISC1 participates in interneuron development and add to our understanding of how DISC1 variants can affect susceptibility to psychiatric illness.