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CRISPR/Cas-based genome editing has dramatically improved genetic modification technology.In-situelectroporation called genome editing via oviductal nucleic acid delivery (GONAD), which eliminates the need forex vivoembryo handling, is technically the simplest method for gene transfer and can be performed in laboratories without developmental engineering expertise. However, the use of this method remains challenging in the case of large-fragment knock-in, such as gene expression cassettes. Adeno-associated viruses (AAV) act as donor DNA for homologous recombination in infectious cells, including rodent embryos. In this study, we demonstrated simultaneous electroporation of AAV donors and CRISPR/Cas9 components into embryos to create knock-in animals, and successfully generated knock-in rats carrying a gene cassette with a length of 3.0 kb using a small number of animals andin-situelectroporation. These findings indicate that this technique is an efficient high-throughput strategy for producing genetically modified rodents and may be applicable to other animal species.

Optogenetic tools such as channelrhodopsin-2 (ChR2) enable the manipulation and mapping of neural circuits. However, ChR2 variants selectively transported down a neuron’s long-range axonal projections for precise presynaptic activation remain lacking. As a result, ChR2 activation is often contaminated by the spurious activation of en passant fibers that compromise the accurate interpretation of functional effects. Here, we explored the engineering of a ChR2 variant specifically localized to presynaptic axon terminals. The metabotropic glutamate receptor 2 (mGluR2) C-terminal domain fused with a proteolytic motif and axon-targeting signal (mGluR2-PA tag) localized ChR2-YFP at axon terminals without disturbing normal transmission. mGluR2-PA-tagged ChR2 evoked transmitter release in distal projection areas enabling lower levels of photostimulation. Circuit connectivity mapping in vivo with the Spike Collision Test revealed that mGluR2-PA-tagged ChR2 is useful for identifying axonal projection with significant reduction in the polysynaptic excess noise. These results suggest that the mGluR2-PA tag helps actuate trafficking to the axon terminal, thereby providing abundant possibilities for optogenetic experiments., Hamada et al. engineer and utilise a channelrhodopsin-2 variant that is localized to presynaptic axon terminals. They demonstrate its use for circuitry mapping in vivo and thus provide a useful tool for future optogenetic experiments

GM1-gangliosidosis is a progressive neurodegenerative glycosphingolipidosis resulting from a

Elucidating neuronal circuitry mechanisms underlying adaptive behaviors and memory formation is fundamental to understanding brain function. Pavlovian fear conditioning studies have provided important insights into the neuronal mechanisms of conditioned and unconditioned stimuli in the amygdala, and their associative mechanisms and plasticity have been intensively studied. Recent advances in optogenetics and pharmacogenetics combined with genetic manipulation tools, however, reveal new insights into unconditioned stimuli themselves. Importantly, how unconditioned stimuli are transmitted and regulated has been explored at the circuitry level. This review focuses on recent advances and earlier studies on the specific neuronal pathways regulating unconditioned stimulus information processing in intra-amygdala and extra-amygdala circuits, and provides a working model of the neural tuning mechanisms underlying adaptive behaviors.

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disease (LSD) caused by a deficiency of the iduronate-2-sulfatase (IDS) that catabolizes glycosaminoglycans (GAGs). Abnormal accumulations of GAGs in somatic cells lead to various manifestations including central nervous system (CNS) disease. Enzyme replacement therapy (ERT) and hematopoietic stem cell transplantation (HSCT) are the currently available therapy for MPS II, but both therapies fail to improve CNS manifestations. We previously showed that hematopoietic stem cell targeted gene therapy (HSC-GT) with lethal irradiation improved CNS involvement in a murine model of MPS II which lacks the gene coding for IDS. However, the strong preconditioning, with lethal irradiation, would cause a high rate of morbidity and mortality. Therefore, we tested milder preconditioning procedures with either low dose irradiation or low dose irradiation plus an anti c-kit monoclonal antibody (ACK2) to assess CNS effects in mice with MPS II after HSC-GT. Mice from all the HSC-GT groups displayed super-physiological levels of IDS enzyme activity and robust reduction of abnormally accumulated GAGs to the wild type mice levels in peripheral organs. However, only the mice treated with lethal irradiation showed significant cognitive function improvement as well as IDS elevation and GAG reduction in the brain. These results suggest that an efficient engraftment of genetically modified cells for HSC-GT requires strong preconditioning to ameliorate CNS involvement in cases with MPS II.

Pogo transposable element derived with ZNF domain (POGZ) has been identified as one of the most recurrently de novo mutated genes in patients with neurodevelopmental disorders (NDDs), including autism spectrum disorder (ASD), intellectual disability and White-Sutton syndrome; however, the neurobiological basis behind these disorders remains unknown. Here, we show that POGZ regulates neuronal development and that ASD-related de novo mutations impair neuronal development in the developing mouse brain and induced pluripotent cell lines from an ASD patient. We also develop the first mouse model heterozygous for a de novo POGZ mutation identified in a patient with ASD, and we identify ASD-like abnormalities in the mice. Importantly, social deficits can be treated by compensatory inhibition of elevated cell excitability in the mice. Our results provide insight into how de novo mutations on high-confidence ASD genes lead to impaired mature cortical network function, which underlies the cellular pathogenesis of NDDs, including ASD., De novo mutations significantly contribute to autism spectrum disorders (ASD). Here, the authors demonstrate that ASD-associated de novo mutations in the POGZ gene, one of a high-confidence ASD gene, lead to ASD-related impaired neuronal development and disrupted mature cortical network function.

The role of the neuropeptide calcitonin gene‐related peptide (CGRP) is well established in nociceptive behaviors. CGRP is highly expressed in the projection pathway from the parabrachial nucleus to the laterocapsular region of the central amygdala (CeC), which plays a critical role in relaying nociceptive information. The CeC is a key structure in pain behavior because it integrates and modulates nociceptive information along with other sensory signals. Previous studies have demonstrated that blockade of the amygdalar CGRP‐signaling cascade attenuates nociceptive behaviors in pain models, while CGRP application facilitates amygdalar synaptic transmission and induces pain behaviors. Despite these lines of evidence, it remains unclear whether endogenous CGRP is involved in the development of nociceptive behaviors accompanied with amygdalar plasticity in a peripheral inflammation model in vivo. To directly address this, we utilized a previously generated CGRP knockout (KO) mouse to longitudinally study formalin‐induced plasticity and nociceptive behavior. We found that synaptic potentiation in the right PB‐CeC pathway that was observed in wild‐type mice was drastically attenuated in the CGRP KO mice 6 h post‐inflammation, when acute nociceptive behavior was no longer observed. Furthermore, the bilateral tactile allodynia 6 h post‐inflammation was significantly decreased in the CGRP KO mice. In contrast, the acute nociceptive behavior immediately after the formalin injection was reduced only at 20–25 min post‐injection in the CGRP KO mice. These results suggest that endogenous CGRP contributes to peripheral inflammation‐induced synaptic plasticity in the amygdala, and this plasticity may underlie the exaggerated nociception–emotion linkage in pain chronification.

A large majority of neurons in the superficial layer of the dorsal horn projects to the lateral parabrachial nucleus (LPB). LPB neurons then project to the capsular part of the central amygdala (CeA; CeC), a key structure underlying the nociception-emotion link. LPB-CeC synaptic transmission is enhanced in various pain models by using electrical stimulation of putative fibers of LPB origin in brain slices. However, this approach has limitations for examining direct monosynaptic connections devoid of directly stimulating fibers from other structures and local GABAergic neurons. To overcome these limitations, we infected the LPB of rats with an adeno-associated virus vector expressing channelrhodopsin-2 and prepared coronal and horizontal brain slices containing the amygdala. We found that blue light stimulation resulted in monosynaptic excitatory postsynaptic currents (EPSCs), with very small latency fluctuations, followed by a large polysynaptic inhibitory postsynaptic current in CeC neurons, regardless of the firing pattern type. Intraplantar formalin injection at 24 h before slice preparation significantly increased EPSC amplitude in late firing-type CeC neurons. These results indicate that direct monosynaptic glutamatergic inputs from the LPB not only excite CeC neurons but also regulate CeA network signaling through robust feed-forward inhibition, which is under plastic modulation in response to persistent inflammatory pain.

Nociceptive signals originating in the periphery are conveyed to the brain through specific afferent and ascending pathways. The spino-(trigemino-)parabrachio-amygdaloid pathway is one of the principal pathways mediating signals from nociception-specific ascending neurons to the central amygdala, a limbic structure involved in aversive signal-associated emotional responses, including the emotional aspects of pain. Recent studies suggest that the right and left central amygdala play distinct roles in the regulation of nociceptive responses. Using a latent formalin inflammatory pain model of the rat, we analyzed the right-left differences in synaptic potentiation at the synapses formed between the fibers from the lateral parabrachial nucleus and central amygdala neurons as well as those in the c-Fos expression in the lateral parabrachial nucleus, central amygdala, and the basolateral/lateral amygdala after formalin injection to either the right or left side of the rat upper lip. Although the single-sided formalin injection caused a significant bilateral increase in c-Fos-expressing neurons in the lateral parabrachial nucleus with slight projection-side dependence, the increase in the amplitude of postsynaptic excitatory currents and the number of c-Fos-expressing neurons in the central amygdala occurred predominantly on the right side regardless of the side of the inflammation. Although there was no significant correlation in the number of c-Fos-expressing neurons between the lateral parabrachial nucleus and central amygdala in the formalin-injected animals, these numbers were significantly correlated between the basolateral amygdala and central amygdala. It is thus concluded that the lateral parabrachial nucleus-central amygdala synaptic potentiation reported in various pain models is not a simple Hebbian plasticity in which raised inputs from the lateral parabrachial nucleus cause lateral parabrachial nucleus-central amygdala potentiation but rather an integrative and adaptive response involving specific mechanisms in the right central amygdala.

Summary Objective Patients with epileptic spasms are at high risk for learning and memory impairment later in life. We examined whether synaptic plasticity is affected in the adult hippocampus, a structure responsible for learning and memory, using an animal model of epileptic spasms of unknown cause. Methods We produced a rat model of N-methyl-d-aspartate (NMDA)–induced spasms combined with prenatal betamethasone administration. In 6- to 11-week-old rats, we evaluated the long-term potentiation (LTP) and general properties of synaptic transmission in pyramidal neurons in the CA1 area of the hippocampus in brain slices. Results The magnitude of LTP by theta burst stimulation was significantly larger in adult rats with a history of infantile NMDA injections than in control rats and rats that received additional adrenocorticotropic hormone (ACTH) treatment. The frequency of spontaneous excitatory transmission, but not inhibitory transmission, was smaller in adult rats with a history of infantile NMDA injections. Significance This study is the first to provide a basis for the alteration of synaptic plasticity and transmission in a model of epileptic spasms of unknown cause. Postnatal NMDA treatment causing epileptic spasms–like aberrant episodes in the early stage of life in rats has a latent influence on various forms of synaptic plasticity in the hippocampus. Our results provide a novel insight into cognitive impairment that appears later in life in patients with a history of epileptic spasms.

Synapses of amphids defective (SAD)-A/B kinases control various steps in neuronal development and differentiation, such as axon specifications and maturation in central and peripheral nervous systems. At mature pre-synaptic terminals, SAD-B is associated with synaptic vesicles and the active zone cytomatrix; however, how SAD-B regulates neurotransmission and synaptic plasticity in vivo remains unclear. Thus, we used SAD-B knockout (KO) mice to study the function of this pre-synaptic kinase in the brain. We found that the paired-pulse ratio was significantly enhanced at Shaffer collateral synapses in the hippocampal CA1 region in SAD-B KO mice compared with wild-type littermates. We also found that the frequency of the miniature excitatory post-synaptic current was decreased in SAD-B KO mice. Moreover, synaptic depression following prolonged low-frequency synaptic stimulation was significantly enhanced in SAD-B KO mice. These results suggest that SAD-B kinase regulates vesicular release probability at pre-synaptic terminals and is involved in vesicular trafficking and/or regulation of the readily releasable pool size. Finally, we found that hippocampus-dependent contextual fear learning was significantly impaired in SAD-B KO mice. These observations suggest that SAD-B kinase plays pivotal roles in controlling vesicular release properties and regulating hippocampal function in the mature brain. Synapses of amphids defective (SAD)-A/B kinases control various steps in neuronal development and differentiation, but their roles in mature brains were only partially known. Here, we demonstrated, at mature pre-synaptic terminals, that SAD-B regulates vesicular release probability and synaptic plasticity. Moreover, hippocampus-dependent contextual fear learning was significantly impaired in SAD-B KO mice, suggesting that SAD-B kinase plays pivotal roles in controlling vesicular release properties and regulating hippocampal function in the mature brain.

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

We found 140 neutrino-induced muons in 854.24 live days in the MINOS far detector. We looked for evidence of neutrino disappearance in this data set by computing the ratio of the number of low momentum muons to the sum of the number of high momentum and unknown momentum muons for both data and Monte Carlo expectation in the absence of neutrino oscillations. The ratio of data and Monte Carlo ratios is consistent with an oscillation signal. A fit to the data for the oscillation parameters excludes the null oscillation hypothesis at the 94% confidence level. We separated the muons by charge sign in both the data and Monte Carlo events and found the ratio of the total number of negative to positive muons in both samples. The ratio of those ratios is a test of CPT conservation. The result is consistent with CPT conservation., 14 pages, 15 figures

This study aims to determine the distribution and frequency of verocytotoxin genes in human faecal clinical isolates of Escherichia coli O157 in Northern Ireland during the period 1997-2000, using a special four-target multiplex polymerase chain reaction (PCR) assay. One hundred and thirty two isolates of E. coli O157:H7 cultured during the four-year period (1997 [n=28]; 1998 [n=25]); 1999 (n=43); 2000 [n=36]), representing approximately 79% of total E. coli O157 laboratory isolations throughout N. Ireland, are examined for the presence of verocytotoxin gene loci (VT1, VT2 and eae) using a multiplex PCR assay. These isolates originate from the four Regional Area Health Boards that constitute the healthcare system in N. Ireland as follows: Eastern (53.8%; n=71), Northern (34.1%; n=45), Western (6.8%; n=9) and Southern (5.3%; n=7). Results showed that over 80% of these isolates possessed the VT2 and eae gene loci, with the remainder being predominantly VT1-, VT2- and eae-positive. None possessed the VT1 gene locus alone. Development and adoption of this simple four-target (three virulence and one control gene loci) multiplex PCR assay and subsequent recording of resulting verocytotoxin-typing data in a database, permitted local, rapid determination of carriage of known molecular virulence determinants of E. coli O157 isolates, which may aid in outbreak-related epidemiological investigations or other longitudinal studies.

Key points • While the metabotropic glutamate receptor (mGluR) is supposed to modulate L-type voltage-dependent calcium channels (L-VDCCs), its reported actions include both facilitation and suppression, and thus the modulation of L-VDCCs by synaptic activity has still been under debate. • In this study, using acute hippocampal slices of subtype-specific knockout mice, we have shown that mGluR5 induces facilitation of the depolarization-evoked calcium current. • This facilitation was not accompanied by the change in single-channel properties of the L-VDCC itself, but required the activation of calcium-induced calcium release that was triggered by L-VDCC opening. • L-VDCCs and mGluR5 were shown to form a complex by coimmunoprecipitation, suggesting that the specific functional coupling between mGluR5, InsP3 receptors and L-VDCCs played a pivotal role in the calcium-current facilitation. • Our study has identified a novel mechanism of the interaction between the mGluR and calcium signalling, and suggested a contribution of mGluR5 to synaptic plasticity. Abstract Activity-dependent regulation of calcium dynamics in neuronal cells can play significant roles in the modulation of many cellular processes such as intracellular signalling, neuronal activity and synaptic plasticity. Among many calcium influx pathways into neurons, the voltage-dependent calcium channel (VDCC) is the major source of calcium influx, but its modulation by synaptic activity has still been under debate. While the metabotropic glutamate receptor (mGluR) is supposed to modulate L-type VDCCs (L-VDCCs), its reported actions include both facilitation and suppression, probably reflecting the uncertainty of both the molecular targets of the mGluR agonists and the source of the recorded calcium signal in previous reports. In this study, using subtype-specific knockout mice, we have shown that mGluR5 induces facilitation of the depolarization-evoked calcium current. This facilitation was not accompanied by the change in single-channel properties of the VDCC itself; instead, it required the activation of calcium-induced calcium release (CICR) that was triggered by VDCC opening, suggesting that the opening of CICR-coupled cation channels was essential for the facilitation. This facilitation was blocked or reduced by the inhibitors of both L-VDCCs and InsP3 receptors (InsP3Rs). Furthermore, L-VDCCs and mGluR5 were shown to form a complex by coimmunoprecipitation, suggesting that the specific functional coupling between mGluR5, InsP3Rs and L-VDCCs played a pivotal role in the calcium-current facilitation. Finally, we showed that mGluR5 enhanced VDCC-dependent long-term potentiation (LTP) of synaptic transmission. Our study has identified a novel mechanism of the interaction between the mGluR and calcium signalling, and suggested a contribution of mGluR5 to synaptic plasticity.

The hippocampus is essential for the formation of certain types of memory, and synaptic plasticity such as long-term potentiation (LTP) is widely accepted as a cellular basis of hippocampus-dependent memory. Although LTP in both perforant path-dentate gyrus (DG) granule cell and CA3-CA1 pyramidal cell synapses is similarly dependent on activation of postsynaptic N-methyl-D-aspartate receptors, several reports suggest that modulation of LTP by γ-aminobutyric acid (GABA) receptor-mediated inhibitory inputs is stronger in perforant path-DG granule cell synapses. However, little is known about how different the mechanism and physiological relevance of the GABAergic modulation of LTP induction are among different brain regions. We confirmed that the action of GABA(A) receptor antagonists on LTP was more prominent in the DG, and explored the mechanism introducing such difference by examining two types of GABA(A) receptor-mediated inhibition, i.e. synaptic and tonic inhibition. As synaptic inhibition, we compared inhibitory vs. excitatory monosynaptic responses and their summation during an LTP-inducing stimulus, and found that the balance of the summated postsynaptic currents was biased toward inhibition in the DG. As tonic inhibition, or sustained activation of extrasynaptic GABA(A) receptors by ambient GABA, we measured the change in holding currents of the postsynaptic cells induced by GABA(A) receptor antagonists, and found that the tonic inhibition was significantly stronger in the DG. Furthermore, we found that tonic inhibition was associated with LTP modulation. Our results suggest that both the larger tonic inhibition and the larger inhibitory/excitatory summation balance during conditioning are involved in the stronger inhibitory modulation of LTP in the DG.

Major depressive and bipolar disorders are serious illnesses that affect millions of people. Growing evidence implicates glutamate signalling in depression, though the molecular mechanism by which glutamate signalling regulates depression-related behaviour remains unknown. In this study, we provide evidence suggesting that tyrosine phosphorylation of the NMDA receptor, an ionotropic glutamate receptor, contributes to depression-related behaviour. The NR2A subunit of the NMDA receptor is tyrosine-phosphorylated, with Tyr 1325 as its one of the major phosphorylation site. We have generated mice expressing mutant NR2A with a Tyr-1325-Phe mutation to prevent the phosphorylation of this site in vivo. The homozygous knock-in mice show antidepressant-like behaviour in the tail suspension test and in the forced swim test. In the striatum of the knock-in mice, DARPP-32 phosphorylation at Thr 34, which is important for the regulation of depression-related behaviour, is increased. We also show that the Tyr 1325 phosphorylation site is required for Src-induced potentiation of the NMDA receptor channel in the striatum. These data argue that Tyr 1325 phosphorylation regulates NMDA receptor channel properties and the NMDA receptor-mediated downstream signalling to modulate depression-related behaviour.

Modern theories on memory storage have mainly focused on Hebbian long-term potentiation (LTP), which requires coincident activation of presynaptic and postsynaptic neurons for its induction. In addition to Hebbian LTP, the roles of non-Hebbian plasticity have also been predicted by some neuronal network models. However, still only a few pieces of evidence have been presented for the presence of such plasticity. In this study, we show in mouse hippocampal slices that LTP can be induced by postsynaptic repetitive depolarization alone in the absence of presynaptic inputs. The induction was dependent on voltage-dependent calcium channels instead of NMDA receptors (NMDARs), whereas the expression mechanism was shared with conventional NMDAR-dependent LTP. During the potentiation, the amplitude of spontaneous EPSCs was increased, suggesting a novel neuron-wide nature of this form of LTP. Furthermore, we also successfully induced LTP with trains of action potentials, which supported the possible existence of depolarizing pulse-induced LTPin vivo. Based on these findings, we suggest a model in which neuron-wide LTP works in concert with synapse-specific Hebbian plasticity to help information processing in memory formation.

Molecular epidemiology of verocytoxigenic Escherichia coli O157:H7 is important to help elucidate reservoirs and modes of transmission, particularly between animals and humans. As the recA gene locus is now beginning to gain application in bacterial genotyping schemes, and as it has not been examined previously in E. coli O157 isolates, this study aims to examine potential polymorphic variation as a possible epidemiological marker for the subspecies characterisation of clinically significant verocytotoxigenic E. coli O157:H7. A novel polymerase chain reaction (PCR) assay was designed to target a 638 bp region of the recA gene in E. coli O157 isolates. The PCR amplification of genomic DNA from extracted organisms was able to generate an amplicon of the expected size (approximately 638 bp) for all E. coli O157:H7 examined (n=80), as well as for other non-O157 E. coli and other members of the Enterobacteriaeceae including Citrobacter, Hafnia, Shigella, Enterobacter and Providencia. Subsequent restriction fragment length polymorphism (RFLP) and single-stranded conformational polymorphism (SSCP) analyses of these recA amplicons were able to differentiate E. coli O157 from the organisms examined, but were unable to distinguish between 79 isolates of wild-type E. coli O157, suggesting a highly conserved recA gene structure within the local population of organisms examined.

The NMDA-type glutamate receptor is a heteromeric complex composed of the NR1 and at least one of the NR2 subunits. Switching from the NR2B to the NR2A subunit is thought to underlie functional alteration of the NMDA receptor during synaptic maturation, and it is generally believed that it results in preferential localization of NR2A subunits on the synaptic site and that of NR2B subunits on the extracellular site in the mature brain. It has also been proposed that activation of the NR2A and NR2B subunits results in long-term potentiation (LTP) and long-term depression (LTD), respectively. Furthermore, recent reports suggest that synaptic and extrasynaptic receptors may have distinct roles in synaptic plasticity as well as in gene expression associated with neuronal death. Here, we have investigated whether NR2B subunit-containing receptors are present and functional at mature synapses in the lateral nucleus of the amygdala (LA) and the CA1 region of the hippocampus, comparing their properties between the two brain regions. We have found, in contrast to the above hypotheses, that the NR2B subunit significantly contributes to synaptic transmission as well as LTP induction. Furthermore, its contribution is greater in the LA than in the CA1 region, and biophysical properties of NMDA receptors and the NR2B/NR2A ratio are different between the two brain regions. These results indicate that NR2B subunit-containing NMDA receptors accumulate on the synaptic site and are responsible for the unique properties of synaptic function and plasticity in the amygdala.

Human cryptosporidiosis has emerged as an important gastrointestinal infection in the 1990s as a result of the ingestion of mainly contaminated water and to a lesser extent foodstuffs containing the protozoan parasite, Cryptosporidium parvum. This pathogen has particular clinical significance for immunocompromised persons, including AIDS patients and cancer patients receiving toxic chemotherapeutic drug regimens. There have been a limited number of studies performed examining the occurrence of the parasite on vegetables, including lettuce. Detection rates are very dependent on the laboratory isolation technique employed and has ranged from 1.2% to 14.5%. Current best practice of laboratory recovery, isolation and detection methods include detergent removal, oocysts concentration by immunomagnetic separation, followed by a combination of immunofluorescent microscopy and a nested PCR approach. Employment of contaminated non-potable water in the production of vegetables, particularly lettuce, may represent an important potential source of entry of pathogens into food processing and the human food chain. Given that lettuce is an important constituent of hamburger dressing, and the size of the fast-food industry, where lettuce is an important constituent, horticultural producers of lettuce should therefore place special emphasis on developing suitable and efficient Hazard Analysis Critical Control Point strategies for the critical control of oocysts depending on the type of unit operation employed and vegetable being processed. This review aims to examine (i) the incidence of C. parvum in vegetables, particularly lettuce and (ii) laboratory detection methods for the isolation and identification of this parasite from lettuce.

Shellfish including oysters (Crassostrea gigas), cockles (Cerastoderma edule) and mussels (Mytilus edulis), have previously been described as an important source of thermophilic campylobacters, with the potential of causing acute bacterial gastroenteritis in humans. Previous genotyping studies employing the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) typing, based on the flagellin (flaA) gene have been unable to generate an amplicon for the urease-positive thermophilic Campylobacter (UPTC), which are the predominant taxa associated with shellfish, largely caused by sequence diversity between the UPTC group and C. jejuni. Hence the aim of this study was to develop a modified PCR-RFLP genotyping assay, employing polymorphisms within the flagellin (flaA) gene of UPTC organisms, which would now allow the successful amplification and typing of previously nontypable UPTC isolates obtained from natural marine environments. A novel primer pair (UPTC flaF/UPTC flaR) was designed based on conserved regions within the flaA gene locus of UPTC organisms to generate a 1,358 bp amplicon for all UPTC organisms tested. RFLP analysis with DdeI in combination with computational analysis of genetic relatedness using BioNumerics software demonstrated the presence of four distinct flaA genotypes, among the seven UPTC isolates. In conclusion, this study describes a PCR-RFLP method, based on modified primers from UPTC flaA gene sequences that may be successfully applied to examine subspecies relatedness of UPTC organisms from natural environments, including shellfish.

Phosphorylation of neural proteins in response to a diverse array of external stimuli is one of the main mechanisms underlying dynamic changes in neural circuitry. The NR2B subunit of the NMDA receptor is tyrosine-phosphorylated in the brain, with Tyr-1472 its major phosphorylation site. Here, we generate mice with a knockin mutation of the Tyr-1472 site to phenylalanine (Y1472F) and show that Tyr-1472 phosphorylation is essential for fear learning and amygdaloid synaptic plasticity. The knockin mice show impaired fear-related learning and reduced amygdaloid long-term potentiation. NMDA receptor-mediated CaMKII signaling is impaired in YF/YF mice. Electron microscopic analyses reveal that the Y1472F mutant of the NR2B subunit shows improper localization at synapses in the amygdala. We thus identify Tyr-1472 phosphorylation as a key mediator of fear learning and amygdaloid synaptic plasticity.

Inhibitors of both phosphatidylinositol-3-kinase (PI3-kinase) and MAPK/ERK (mitogen-activate protein kinase/extracellular signal-related kinase) activation inhibit NMDA receptor-dependent long-term potentiation (LTP). PI3-kinase inhibitors also block activation of ERK by NMDA receptor stimulation, suggesting that PI3-kinase inhibitors block LTP because PI3-kinase is an essential upstream regulator of ERK activation. To examine this hypothesis, we investigated the effects of PI3-kinase inhibitors on ERK activation and LTP induction in the CA1 region of mouse hippocampal slices. Consistent with the notion that ERK activation by NMDA receptor stimulation is PI3-kinase dependent, the PI3-kinase inhibitor wortmannin partially inhibited ERK2 activation induced by bath application of NMDA and strongly suppressed ERK2 activation by high-frequency synaptic stimulation. PI3-kinase and MEK (MAP kinase kinase) inhibitors had very different effects on LTP, however. Both types of inhibitors suppressed LTP induced by theta-frequency trains of synaptic stimulation, but only PI3-kinase inhibitors suppressed the induction of LTP by high-frequency stimulation or low-frequency stimulation paired with postsynaptic depolarization. Concentrations of PI3-kinase inhibitors that inhibited LTP when present during high-frequency stimulation had no effect on potentiated synapses when applied after high-frequency stimulation, suggesting that PI3-kinase is specifically involved in the induction of LTP. Finally, we found that LTP induced by theta-frequency stimulation was MEK inhibitor insensitive but still PI3-kinase dependent in hippocampal slices from PSD-95 (postsynaptic density-95) mutant mice. Together, our results indicate that the role of PI3-kinase in LTP is not limited to its role as an upstream regulator of MAPK signaling but also includes signaling through ERK-independent pathways that regulate LTP induction.

M. W ATABE, J .R. R AO, T .A. S TEWART, J . X U, B .C. M ILLAR, L . X IAO, C .J. L OWERY, J .S.G. D OOLEY A N D J . E . M O O R E . 2003. Aims: To examine the prevalence and diversity of bacterial faecal pathogens in unseparated slurry, separated solids and liquid fractions from a commercial pig farm. Methods: A total of 43 stored slurry specimens originating from a fattening house over the period February–April 2002 were analysed, consisting of unseparated (n ¼ 14) slurry, separated solids (n ¼ 16) and separated liquid (n ¼ 13). Specimens were examined for the presence of five bacterial pathogens including Salmonella spp., Shigella spp., Campylobacter spp., Escherichia coli O157 and Yersinia enterocolitica. Selective enrichment and plating methods were employed for detection of Salmonella spp. and Campylobacter spp. and conventional selective plating techniques for the remaining genera. Antibiogram profiles to 12 antibiotic agents were obtained for all Salmonella isolates obtained. Results: Salmonella spp. were identified in all components of the slurry specimens, whereas Campylobacter spp. was only recovered from the unseparated and separated liquid fractions. In both cases, the separated liquid fraction had the highest prevalence of pathogens and the separated solid fraction had the lowest prevalence. None of the slurry specimens examined were positive for E. coli O157:H7, Shigella spp. or Y. enterocolitica. Twentynine isolates of Salmonella were recovered from the slurry specimens, comprising seven serovars, of which Salmonella manhattan was the most prevalent, accounting for over half [15 of 29 (51AE7%)] of all Salmonella isolates. Salmonella anatum, Salm. derby, Salm. give, Salm. heidelberg, Salm. simi and Salm. stanley serovars were also recovered. All Salmonella isolates were sensitive to ampicillin, augmentin (amoxicillin/clavulanic acid), chloramphenicol, ciprofloxacin, gentamicin, kanamycin and trimethoprim, but has variable resistance to tetracycline (100%), sulphonamides (84AE6%), furazolidone (38AE5%), nalidixic acid (15AE4%) and streptomycin (15AE4%). The majority (57AE7%) of isolates displayed antibiotic resistance to at least two antibiotic agents, followed by 34AE6% of isolates being resistant to three agents and the remainder (7AE7%) being resistant to four antibiotics. Significance and Impact of the Study: This study demonstrated a marked reduction in the prevalence of Campylobacter and Salmonella in the solids component of separated pig slurry. The adoption of control processes such as aeration of slurry prior to its spread onto agricultural land and newer approaches to pathogen reduction should be investigated, to reduce the transmission of pathogens from pig slurry to the environment.

At excitatory synapses, the postsynaptic scaffolding protein postsynaptic density 95 (PSD-95) couples NMDA receptors (NMDARs) to the Ras GTPase-activating protein SynGAP. The close association of SynGAP and NMDARs suggests that SynGAP may have an important role in NMDAR-dependent activation of Ras signaling pathways, such as the MAP kinase pathway, and in synaptic plasticity. To explore this issue, we examined long-term potentiation (LTP), p42 MAPK (ERK2) signaling, and spatial learning in mice with a heterozygous null mutation of the SynGAP gene (SynGAP(−/+)). In SynGAP(−/+) mutant mice, the induction of LTP in the hippocampal CA1 region was strongly reduced in the absence of any detectable alteration in basal synaptic transmission and NMDAR-mediated synaptic currents. Although basal levels of activated ERK2 were elevated in hippocampal extracts from SynGAP(−/+)mice, NMDAR stimulation still induced a robust increase in ERK activation in slices from SynGAP(−/+) mice. Thus, although SynGAP may regulate the ERK pathway, its role in LTP most likely involves additional downstream targets. Consistent with this, the amount of potentiation induced by stimulation protocols that induce an ERK-independent form of LTP were also significantly reduced in slices from SynGAP(−/+) mice. An elevation of basal phospho-ERK2 levels and LTP deficits were also observed in SynGAP(−/+)/H-Ras(−)(/)(−)double mutants, suggesting that SynGAP may normally regulate Ras isoforms other than H-Ras. A comparison of SynGAP and PSD-95 mutants suggests that PSD-95 couples NMDARs to multiple downstream signaling pathways with very different roles in LTP and learning.

Activation of metabotropic glutamate receptors (mGluRs) with the group I mGluR selective agonist (R,S)-3,5-dihydroxyphenylglycine (DHPG) induces a long-term depression (LTD) of excitatory synaptic transmission in the CA1 region of the hippocampus. Here we investigated the potential roles of pre- and postsynaptic processes in the DHPG-induced LTD at excitatory synapses onto hippocampal pyramidal cells in the mouse hippocampus. Activation of mGluRs with DHPG, but not ACPD, induced LTD at both Schaffer collateral/commissural fiber synapses onto CA1 pyramidal cells and at associational/commissural fiber synapses onto CA3 pyramidal cells. DHPG-induced LTD was blocked when the G-protein inhibitor guanosine-5′- O-(2-thiodiphosphate) was selectively delivered into postsynaptic CA1 pyramidal cells via an intracellular recording electrode, suggesting that DHPG depresses synaptic transmission through a postsynaptic, GTP-dependent signaling pathway. The effects of DHPG were also strongly modulated, however, by experimental manipulations that altered presynaptic calcium influx. In these experiments, we found that elevating extracellular Ca2+ concentrations ([Ca2+]o) to 6 mM almost completely blocked the effects of DHPG, whereas lowering [Ca2+]o to 1 mM significantly enhanced the ability of DHPG to depress synaptic transmission. Enhancing Ca2+ influx by prolonging action potential duration with bath applications of the K+ channel blocker 4-aminopyridine (4-AP) also strongly reduced the effects of DHPG in the presence of normal [Ca2+]o (2 mM). Although these findings indicate that alterations in Ca2+-dependent signaling processes strongly regulate the effects of DHPG on synaptic transmission, they do not distinguish between potential pre- versus postsynaptic sites of action. We found, however, that while inhibiting both pre- and postsynaptic K+ channels with bath-applied 4-AP blocked the effects of DHPG; inhibition of postsynaptic K+channels alone with intracellular Cs+ and TEA had no effect on the ability of DHPG to inhibit synaptic transmission. This suggests that presynaptic changes in transmitter release contribute to the depression of synaptic transmission by DHPG. Consistent with this, DHPG induced a persistent depression of both AMPA and N-methyl-d-aspartate receptor-mediated components of excitatory postsynaptic currents in voltage-clamped pyramidal cells. Together our results suggest that activation of postsynaptic mGluRs suppresses transmission at excitatory synapses onto CA1 pyramidal cells through presynaptic effects on transmitter release.

The N-methyl-d-aspartate receptor (NMDAR) plays various physiological and pathological roles in neural development, synaptic plasticity and neuronal cell death. It is composed of two GluN1 and two GluN2 subunits and, in the neonatal hippocampus, most synaptic NMDARs are GluN2B-containing receptors, which are gradually replaced with GluN2A-containing receptors during development. Here, we examined whether GluN2A could be substituted for GluN2B in neural development and functions by analysing knock-in (KI) mice in which GluN2B is replaced with GluN2A. The KI mutation was neonatally lethal, although GluN2A-containing receptors were transported to the postsynaptic membrane even without GluN2B and functional at synapses of acute hippocampal slices of postnatal day 0, indicating that GluN2A-containing NMDARs could not be substituted for GluN2B-containing NMDARs. Importantly, the synaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) subunit GluA1 was increased, and the transmembrane AMPAR regulatory protein, which is involved in AMPAR synaptic trafficking, was increased in KI mice. Although the regulation of AMPARs by GluN2B has been reported in cultured neurons, we showed here that AMPAR-mediated synaptic responses were increased in acute KI slices, suggesting differential roles of GluN2A and GluN2B in AMPAR expression and trafficking in vivo. Taken together, our results suggest that GluN2B is essential for the survival of animals, and that the GluN2B–GluN2A switching plays a critical role in synaptic integration of AMPARs through regulation of GluA1 in the whole animal.

Interactions between noradrenergic and cholinergic receptor signaling may be important in some forms of learning. To investigate whether noradrenergic and cholinergic receptor interactions regulate forms of synaptic plasticity thought to be involved in memory formation, we examined the effects of concurrent beta-adrenergic and cholinergic receptor activation on the induction of long-term potentiation (LTP) in the hippocampal CA1 region. Low concentrations of the beta-adrenergic receptor agonist isoproterenol (ISO) and the cholinergic receptor agonist carbachol had no effect on the induction of LTP by a brief train of 5 Hz stimulation when applied individually but dramatically facilitated LTP induction when coapplied. Although carbachol did not enhance ISO-induced increases in cAMP, coapplication of ISO and carbachol synergistically activated p42 mitogen-activated protein kinase (p42 MAPK). This suggests that concurrent beta-adrenergic and cholinergic receptor activation enhances LTP induction by activating MAPK and not by additive or synergistic effects on adenylyl cyclase. Consistent with this, blocking MAPK activation with MEK inhibitors suppressed the facilitation of LTP induction produced by concurrent beta-adrenergic and cholinergic receptor activation. Although MEK inhibitors also suppressed the induction of LTP by a stronger 5 Hz stimulation protocol that induced LTP in the absence of ISO and carbachol, they had no effect on LTP induced by high-frequency synaptic stimulation or low-frequency synaptic stimulation paired with postsynaptic depolarization. Our results indicate that MAPK activation has an important, modulatory role in the induction of LTP and suggest that coactivation of noradrenergic and cholinergic receptors regulates LTP induction via convergent effects on MAPK.

Long-term potentiation (LTP), a persistent enhancement of synaptic transmission that may be involved in some forms of learning and memory, is induced at excitatory synapses in the CA1 region of the hippocampus by coincident presynaptic and postsynaptic activity. Although action potentials back-propagating into dendrites of hippocampal pyramidal cells provide sufficient postsynaptic activity to induce LTP under somein vitroconditions, it is not known whether LTP can be induced by patterns of postsynaptic action potential firing that occur in these cellsin vivo. Here we report that a characteristicin vivopattern of action potential generation in CA1 pyramidal cells known as the complex spike burst enables the induction of LTP during theta frequency synaptic stimulation in the CA1 region of hippocampal slices maintainedin vitro. Our results suggest that complex spike bursting may have an important role in synaptic processes involved in learning and memory formation, perhaps by producing a highly sensitive postsynaptic state during which even low frequencies of presynaptic activity can induce LTP.

It is currently not known whether caffeine has an enhancing effect on long-term memory in humans. We used post-study caffeine administration to test its effect on memory consolidation using a behavioral discrimination task. Caffeine enhanced performance 24 h after administration according to an inverted U-shaped dose-response curve; this effect was specific to consolidation and not retrieval. We conclude that caffeine enhanced consolidation of long-term memories in humans.

Background Pavlovian fear conditioning is a classical form of associative learning, which depends on associative synaptic plasticity in the amygdala. Recent findings suggest that the central amygdala (CeA) plays an active role in the acquisition of fear learning. However, little is known about the synaptic properties of the CeA in fear learning. The capsular part of the central amygdala (CeC) receives direct nociceptive information from the external part of the lateral parabrachial nucleus (lPB), as well as highly processed polymodal signals from the basolateral nucleus of the amygdala (BLA). Therefore, we focused on CeC as a convergence point for polymodal BLA signals and nociceptive lPB signals, and explored the synaptic regulation of these pathways in fear conditioning. Results In this study, we show that fear conditioning results in synaptic potentiation in both lPB-CeC and BLA-CeC synapses. This potentiation is dependent on associative fear learning, rather than on nociceptive or sensory experience, or fear memory retrieval. The synaptic weight of the lPB-CeC and BLA-CeC pathways is correlated in fear-conditioned mice, suggesting that fear learning may induce activity-dependent heterosynaptic interactions between lPB-CeC and BLA-CeC pathways. This synaptic potentiation is associated with both postsynaptic and presynaptic changes in the lPB-CeC and BLA-CeC synapses. Conclusions These results indicate that the CeC may provide an important locus of Pavlovian association, integrating direct nociceptive signals with polymodal sensory signals. In addition to the well-established plasticity of the lateral amygdala, the multi-step nature of this association system contributes to the highly orchestrated tuning of fear learning.

A novel extracellular heat-sensitive inhibitory effect of wild-type cultures of Paenibacillus lentimorbus isolated from mushroom compost upon the in vitro growth of Listeria ivanovii was observed.

Activity-dependent regulation of calcium dynamics in neuronal cells can play significant roles in the modulation of many cellular processes such as intracellular signalling, neuronal activity and synaptic plasticity. Among many calcium influx pathways into neurons, the voltage-dependent calcium channel (VDCC) is the major source of calcium influx, but its modulation by synaptic activity has still been under debate. While the metabotropic glutamate receptor (mGluR) is supposed to modulate L-type VDCCs (L-VDCCs), its reported actions include both facilitation and suppression, probably reflecting the uncertainty of both the molecular targets of the mGluR agonists and the source of the recorded calcium signal in previous reports. In this study, using subtype-specific knockout mice, we have shown that mGluR5 induces facilitation of the depolarization-evoked calcium current. This facilitation was not accompanied by the change in single-channel properties of the VDCC itself; instead, it required the activation of calcium-induced calcium release (CICR) that was triggered by VDCC opening, suggesting that the opening of CICR-coupled cation channels was essential for the facilitation. This facilitation was blocked or reduced by the inhibitors of both L-VDCCs and InsP3 receptors (InsP3Rs). Furthermore, L-VDCCs and mGluR5 were shown to form a complex by coimmunoprecipitation, suggesting that the specific functional coupling between mGluR5, InsP3Rs and L-VDCCs played a pivotal role in the calcium-current facilitation. Finally, we showed that mGluR5 enhanced VDCC-dependent long-term potentiation (LTP) of synaptic transmission. Our study has identified a novel mechanism of the interaction between the mGluR and calcium signalling, and suggested a contribution of mGluR5 to synaptic plasticity.

The hippocampus is essential for the formation of certain types of memory, and synaptic plasticity such as long-term potentiation (LTP) is widely accepted as a cellular basis of hippocampus-dependent memory. Although LTP in both perforant path-dentate gyrus (DG) granule cell and CA3-CA1 pyramidal cell synapses is similarly dependent on activation of postsynaptic N-methyl-D-aspartate receptors, several reports suggest that modulation of LTP by γ-aminobutyric acid (GABA) receptor-mediated inhibitory inputs is stronger in perforant path-DG granule cell synapses. However, little is known about how different the mechanism and physiological relevance of the GABAergic modulation of LTP induction are among different brain regions. We confirmed that the action of GABA(A) receptor antagonists on LTP was more prominent in the DG, and explored the mechanism introducing such difference by examining two types of GABA(A) receptor-mediated inhibition, i.e. synaptic and tonic inhibition. As synaptic inhibition, we compared inhibitory vs. excitatory monosynaptic responses and their summation during an LTP-inducing stimulus, and found that the balance of the summated postsynaptic currents was biased toward inhibition in the DG. As tonic inhibition, or sustained activation of extrasynaptic GABA(A) receptors by ambient GABA, we measured the change in holding currents of the postsynaptic cells induced by GABA(A) receptor antagonists, and found that the tonic inhibition was significantly stronger in the DG. Furthermore, we found that tonic inhibition was associated with LTP modulation. Our results suggest that both the larger tonic inhibition and the larger inhibitory/excitatory summation balance during conditioning are involved in the stronger inhibitory modulation of LTP in the DG.