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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.

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.

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.

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.

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.

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.

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.

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.

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.

An antibody reacting with extracellular polysaccharide from Xanthomonas oryzae pv. oryzae was used to detect the extracellular polysaccharide in infected leaf tissues of rice plants (Oryzae sativa cv. IR24) by immunofluorescence and immunoelectron microscopy. Using extracellular polysaccharide as a hapten and bovine serum albumin as a carrier, an extracellular polysaccharide – bovine serum albumin conjugate was prepared. After a rabbit was immunized against the conjugate, an antibody reacting with extracellular polysaccharide from X. oryzae pv. oryzae was isolated and purified. Using ELISA, the antibody was capable of detecting extracellular polysaccharide at concentrations above 0.1 μg/mL. Immunofluorescent antibody staining of infected rice leaves showed that extracellular polysaccharide produced by X. oryzae pv. oryzae was distributed in both xylem vessels and transverse veins but not in either sieve tubes or mesophyll tissues. This demonstrates that the distribution of extracellular polysaccharide coincided with that of bacteria in the infected leaf tissues. Immunoelectron microscopy showed that localization of extracellular polysaccharide was restricted to the area close to bacterial cells. The amount of extracellular polysaccharide may not be enough to plug xylem vessels of rice leaves infected with X. oryzae pv. oryzae.Key words: anti extracellular polysaccharide antibody, Xanthomonas oryzae pv. oryzae, immunofluorescent microscopy, immunoelectron microscopy, rice plant.

Background The visceral afferents from various cervico-abdominal sensory receptors project to the dorsal vagal complex (DVC), which is composed of the nucleus of the solitary tract (NTS), the area postrema and the dorsal motor nucleus of the vagus nerve (DMX), via the vagus and glossopharyngeal nerves and then the solitary tract (TS) in the brainstem. While the excitatory transmission at the TS-NTS synapses shows strong frequency-dependent suppression in response to repeated stimulation of the afferents, the frequency dependence and short-term plasticity at the TS-DMX synapses, which also transmit monosynaptic information from the visceral afferents to the DVC neurons, remain largely unknown. Results Recording of the EPSCs activated by paired or repeated TS stimulation in the brainstem slices of rats revealed that, unlike NTS neurons whose paired-pulse ratio (PPR) is consistently below 0.6, the distribution of the PPR of DMX neurons shows bimodal peaks that are composed of type I (PPR, 0.6-1.5; 53% of 120 neurons recorded) and type II (PPR, < 0.6; 47%) neurons. Some of the type I DMX neurons showed paired-pulse potentiation. The distinction of these two types depended on the presynaptic release probability and the projection target of the postsynaptic cells; the distinction was not dependent on the location or soma size of the cell, intensity or site of the stimulation, the latency, standard deviation of latency or the quantal size. Repeated stimulation at 20 Hz resulted in gradual and potent decreases in EPSC amplitude in the NTS and type II DMX neurons, whereas type I DMX neurons displayed only slight decreases, which indicates that the DMX neurons of this type could be continuously activated by repeated firing of primary afferent fibers at a high (~10 Hz) frequency. Conclusions These two general types of short-term plasticity might contribute to the differential activation of distinct vago-vagal reflex circuits, depending on the firing frequency and type of visceral afferents.

A genus-specific polymerase chain reaction (PCR)-based assay is developed for the detection and identification of clinically relevant Fusobacterium species, including F. nucleatum and F. necrophorum. Two 16S ribosomal DNA (rDNA) primers, FUSO1 (forward primer: 5'-GAG AGA GCT TTG CGT CC-3' [17-mer]) and FUSO 2 (reverse primer: 5'-TGG GCG CTG AGG TTC GAC -3' [18-mer]) are designed to target conserved regions of the 16S rDNA gene for Fusobacterium spp. Subsequent proof-of-principle studies employing this assay detected Fusobacterium spp. in the faeces of eight (10%) out of 80 patients with suspected gastrointestinal infection. This assay may be used for the genus-specific detection of Fusobacterium spp. from clinical specimens and for subsequent species identification.

A small study was undertaken to examine the microbiological characteristics of spent mushroom compost (SMC), which is the major waste by-product of the mushroom industry and which is regularly disposed off by application to agricultural land. The primary aim of this study was to examine SMC for the presence of faecal bacterial pathogens, including Campylobacter spp., Salmonella spp. and Listeria monocytogenes. Secondly it was desirable to quantify bacterial and fungal populations within SMC, and also qualitatively identify the diversity of bacterial populations within SMC, through employment of rDNA PCR and direct sequencing techniques on the culturable microflora. Conventional microbiological analyses of SMC material (n=30) from six commercial operations in both Northern Ireland and the Republic of Ireland, failed to detect Salmonella spp, Listeria spp. or Campylobacter spp. in any of the SMC material examined. Total aerobic plate counts gave a mean count of log10 7.01 colony forming units (cfu) per gram SMC material (range: log10 6.53-7.52 cfu/g). Fungal counts gave a mean count of log(10) 4.57 cfu per gram SMC material (range: log10 3.93-4.98 cfu/g). From a total of greater than 50 colony picks, a total of 12 bacterial morphotypes were identified and were further examined by employment of partial 16S rRNA gene amplification and sequencing techniques, yielding several genera and species, including Bacillus licheniformis, Bacillus subtilis, Klebsiella/Enterobacter sp. Microbacterium sp. Paenibacillus lentimorbus, Pseudomonas mevalonii, Sphingobacterium multivorum and Stenotrophomonas sp. This is the first preliminary report on the microbial diversity of SMC waste and demonstrates the presence of several species that have not been previously described in SMC, in addition to two potentially novel species within the genera Microbacterium and Stenotrophomonas. It is thereby important to examine the ecological microbe-microbe and plant-microbe interactions that are occurring between the native bacterial soil flora and those added annually (theoretically estimated at approximately 10(18) cells) through the application of SMC. Such studies would be beneficial in helping to ascertain the ecological consequences involved in the disposal of SMC waste on agricultural land.

N-Methyl-d-aspartate (NMDA) receptors regulate structural plasticity by modulating actin organization within dendritic spines. Herein, we report identification and characterization of p250GAP, a novel GTPase-activating protein for Rho family proteins that interacts with the GluRϵ2 (NR2B) subunit of NMDA receptors in vivo. The p250GAP mRNA was enriched in brain, with high expression in cortex, corpus striatum, hippocampus, and thalamus. Within neurons, p250GAP was highly concentrated in the postsynaptic density and colocalized with the GluRϵ2 (NR2B) subunit of NMDA receptors and with postsynaptic density-95. p250GAP promoted GTP hydrolysis of Cdc42 and RhoA in vitro and in vivo. When overexpressed in neuroblastoma cells, p250GAP suppressed the activities of Rho family proteins, which resulted in alteration of neurite outgrowth. Finally, NMDA receptor stimulation led to dephosphorylation and redistribution of p250GAP in hippocampal slices. Together, p250GAP is likely to be involved in NMDA receptor activity-dependent actin reorganization in dendritic spines.

Several signaling mechanisms that are crucial for the induction of long-term potentiation (LTP) by theta frequency (5 Hz) trains of synaptic stimulation are altered in aged animals. To determine whether theta frequency stimulation-induced LTP is particularly sensitive to changes in synaptic function that occur in aged animals, we examined whether activating synapses in the hippocampal CA1 region with three different trains of theta pulse stimulation (TPS) has different effects on synaptic strength in hippocampal slices from aged and young mice. Our results indicate that TPS-induced LTP is not diminished in the aged hippocampus but does show a dependence on L-type voltage-sensitive calcium channels that is not seen in young animals. Moreover, we found that the facilitation of TPS-induced LTP by co-activation of β-adrenergic and cholinergic receptors is strongly reduced in slices from aged animals. Together, these results suggest that memory impairments in aged animals might arise, at least in part, from deficits in the ability of modulatory transmitters to regulate activity-dependent forms of synaptic plasticity.

Using Down syndrome as a model for complex trait analysis, we sought to identify loci from chromosome 21q22.2 which, when present in an extra dose, contribute to learning abnormalities. We generated low-copy-number transgenic mice, containing four different yeast artificial chromosomes (YACs) that together cover approximately 2 megabases (Mb) of contiguous DNA from 21q22,2. We subjected independent lines derived from each of these YAC transgenes to a series of behavioural and learning assays. Two of the four YACs caused defects in learning and memory in the transgenic animals, while the other two YACs had no effect. The most severe defects were caused by a 570-kb YAC; the interval responsible for these defects was narrowed to a 180-kb critical region as a consequence of YAC fragmentation. This region contains the human homologue of a Drosophila gene, minibrain, and strongly implicates it in learning defects associated with Down syndrome.

Background Retinoid signaling pathways mediated by retinoic acid receptor (RAR)/retinoid × receptor (RXR)-mediated transcription play critical roles in hippocampal synaptic plasticity. Furthermore, recent studies have shown that treatment with retinoic acid alleviates age-related deficits in hippocampal long-term potentiation (LTP) and memory performance and, furthermore, memory deficits in a transgenic mouse model of Alzheimer's disease. However, the roles of the RAR/RXR signaling pathway in learning and memory at the behavioral level have still not been well characterized in the adult brain. We here show essential roles for RAR/RXR in hippocampus-dependent learning and memory. In the current study, we generated transgenic mice in which the expression of dominant-negative RAR (dnRAR) could be induced in the mature brain using a tetracycline-dependent transcription factor and examined the effects of RAR/RXR loss. Results The expression of dnRAR in the forebrain down-regulated the expression of RARβ, a target gene of RAR/RXR, indicating that dnRAR mice exhibit dysfunction of the RAR/RXR signaling pathway. Similar with previous findings, dnRAR mice displayed impaired LTP and AMPA-mediated synaptic transmission in the hippocampus. More importantly, these mutant mice displayed impaired hippocampus-dependent social recognition and spatial memory. However, these deficits of LTP and memory performance were rescued by stronger conditioning stimulation and spaced training, respectively. Finally, we found that pharmacological blockade of RARα in the hippocampus impairs social recognition memory. Conclusions From these observations, we concluded that the RAR/RXR signaling pathway greatly contributes to learning and memory, and LTP in the hippocampus in the adult brain.

The Hepatocyte growth factor is the most potent mitogen for hepatocytes in primary culture and is involved in liver regeneration. The expression of the gene appears to be tightly controlled by various humoral factors. To understand the molecular mechanism of the gene expression, we cloned and determined the nucleotide sequence of the 5′-flanking region of the gene. In this region, there are sequences homologous to responding elements of P53, Rb, IL-1, IL-6, glucocorticoids, TPA and TGF-β. We also identified three major transcriptional initiation sites by primer extension analysis of this region. Functional analyses of this region by constructing CAT reporter plasmids indicate that the sequence functions in a tissue specific manner and there is a negative regulatory region which suppresses the gene expression in rat transformed kidney cells.

Background Painful diabetic neuropathy (PDN) is a serious complication of diabetes mellitus that affects a large number of patients in many countries. The molecular mechanisms underlying the exaggerated nociception in PDN have not been established. Recently, duloxetine (DLX), a serotonin and noradrenaline re-uptake inhibitor, has been recommended as one of the first-line treatments of PDN in the United States Food and Drug Administration, the European Medicines Agency and the Japanese Guideline for the Pharmacologic Management of Neuropathic pain. Because selective serotonin re-uptake inhibitors show limited analgesic effects in PDN, we examined whether the potent analgesic effect of DLX contributes toward improving the pathologically aberrant noradrenaline homeostasis in diabetic models. Results In streptozotocin (STZ) (50 mg/kg, i.v.)-induced diabetic rats that exhibited robust mechanical allodynia and thermal hyperalgesia, DLX (10 mg/kg, i.p.) significantly and markedly increased the nociceptive threshold. The analgesic effect of DLX was nullified by the prior administration of N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) (50 mg/kg, i.p.), which drastically eliminated dopamine-beta-hydroxylase- and norepinephrine transporterimmunopositive fibers in the lumbar spinal dorsal horn and significantly reduced the noradrenaline content in the lumbar spinal cord. The treatment with DSP-4 alone markedly lowered the nociceptive threshold in vehicle-treated non-diabetic rats; however, this pro-nociceptive effect was occluded in STZ-treated diabetic rats. Furthermore, STZ-treated rats exhibited a higher amount of dopamine-beta-hydroxylase- and norepinephrine transporterimmunopositive fibers in the dorsal horn and noradrenaline content in the spinal cord compared to vehicle-treated rats. Conclusions Impaired noradrenaline-mediated regulation of the spinal nociceptive network might underlie exaggerated nociception in PDN. DLX might exert its analgesic effect by selective enhancement of noradrenergic signals, thus counteracting this situation.