Behavioral, metabolic, and lipidomic characterization of the 5xFADxTg30 mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is associated with both extracellular amyloid-ß (Aß) plaques and intracellular tau-containing neurofibrillary tangles (NFT). We characterized the behavioral, metabolic and lipidomic phenotype of the 5xFADxTg30 mouse model which contains overexpression of both Aß and tau. Our results independently reproduce several phenotypic traits described previously for this model, while providing additional characterization. This model develops many aspects associated with AD including frailty, decreased survival, initiation of aspects of cognitive decline and alterations to specific lipid classes and molecular lipid species in the plasma and brain. Notably, some sex-specific differences exist in this model and motor impairment with aging in this model does compromise the utility of the model for some movement-based behavioral assessments of cognitive function. These findings provide a reference for individuals interested in using this model to understand the pathology associated with elevated Aß and tau or for testing potential therapeutics for the treatment of AD.

Cortical axon sub-population maintains density, but not turnover, of en passant boutons in the aged APP/PS1 amyloidosis model.

Synaptic dysfunction is one of the key mechanisms associated with cognitive deficits observed in Alzheimer's disease (AD), yet little is known about the presynaptic axonal boutons in AD. Focusing on cortical en passant boutons (EPBs) along axons located in the motor, sensory and prefrontal regions of the cerebral cortex in the APP/PS1 mouse model of AD, we investigated structural properties of EPBs over the lifespan and in response to a midlife environmental enrichment (EE) intervention. At 3, 12, and 18-22 months and following 6 months of midlife EE, we found that EPBs showed remarkable resilience in preserving overall synaptic output, as evidenced by the maintained density of EPBs along the axon shaft across all experimental conditions. Using cranial window imaging to monitor synaptic changes in real time, we report that despite maintaining a stable synaptic density, the dynamic fraction (gains and losses) of EPBs was significantlyreduced at 10-13 months of age in APP/PS1 axons compared to age matched controls.

Associations of Later-Life Education, the BDNF Val66Met Polymorphism and Cognitive Change in Older Adults.

In 358 participants of the Tasmanian Healthy Brain Project, we quantified the cognitive consequences of engaging in varying loads of university-level education in later life, and investigated whether or not BDNF Val66Met affected outcomes. Assessment of neuropsychological, health, and psychosocial function was undertaken at baseline, 12-month, and 24-month follow-up. Education load was positively associated with change in language processing performance, but this effect did not reach statistical significance (P = 0.064). The BDNF Val66Met polymorphism significantly moderated the extent to which education load was associated with improved language processing (P = 0.026), with education load having a significant positive relationship with cognitive change in BDNF Met carriers but not in BDNF Val homozygotes. In older adults who carry BDNF Met, engaging in university-level education improves language processing performance in a load-dependent manner.

Alterations in neurofilaments and the transformation of the cytoskeleton in axons may provide insight into the aberrant neuronal changes of Alzheimer's disease.

Neurofilaments are major protein constituents of the brain, but are particularly abundant in specific subpopulations of neurons and likely have a key role in the regulation of axonal calibre. Neurofilament proteins may also be involved in the transformation of the neuronal cytoskeleton leading to substantial tau pathology in axons damaged by Aß, subsequently leading to neurofibrillary pathology in their cell bodies of origin. An understanding of neurofilamentous changes in axons and subsequent tau pathology may provide insight into how Aß pathology may stimulate an aberrant plasticity-related response of damaged neurons, leading to the progressive and degenerative changes in the neuronal cytoskeleton that result in synapse loss and neuronal degeneration.

ALS-associated mutant FUS inhibits macroautophagy which is restored by overexpression of Rab1.

Amyotrophic lateral sclerosis (ALS) is characterised by the formation of intracellular misfolded protein inclusions that form in motor neurons. Autophagy is the major degradation pathway for aggregate-prone proteins within lysosomes. Autophagy begins by the production of the omegasome, forming the autophagosome membrane, which then fuses with the lysosome. Mutations in fused in sarcoma (FUS) cause 5% of familial ALS cases and FUS-positive inclusions are also formed in sporadic ALS tissues. In this study, we demonstrate that the expression of ALS-associated mutant FUS impairs autophagy in neuronal cells. In mutant FUS-expressing neuronal cells, accumulation of ubiquitinated proteins and autophagy substrates p62 and NBR1 was detected, and formation of both the omegasome and autophagosome was inhibited in these cells. However, overexpression of Rab1 rescued these defects, suggesting that Rab1 is protective in ALS. The number of LC3-positive vesicles was also increased in motor neurons from the spinal cord of an ALS patient carrying a FUS (R521C) mutation compared with a control patient, providing additional evidence that autophagy is dysregulated in mutant FUS-associated ALS. This study provides further understanding of the intricate autophagy system and neurodegeneration in ALS.

Chronic tooth pulp inflammation induces persistent expression of phosphorylated ERK (pERK) and phosphorylated p38 (pp38) in trigeminal subnucleus caudalis.

BACKGROUND: Extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase are transiently phosphorylated (activated) in the spinal cord and trigeminal nucleus by acute noxious stimuli. Acute stimulation of dental pulp induces short-lived ERK activation in trigeminal subnucleus caudalis (Vc), and p38 inhibition attenuates short-term sensitization in Vc induced by acute pulpal stimulation. We have developed a model to study central changes following chronic inflammation of dental pulp that induces long-term sensitization. Here, we examine the effects of chronic inflammation and acute stimulation on the expression of phosphorylated ERK (pERK), phosphorylated p38 (pp38) and Fos in Vc. RESULTS: Chronic inflammation alone induced bilateral expression of pERK and pp38 in Vc, but did not induce Fos expression. Stimulation of both non-inflamed and inflamed pulps significantly increased pERK and pp38 bilaterally; expression was greatest in inflamed, stimulated animals, and was similar following 10-min and 60-min stimulation. Stimulation for 60 min, but not 10 min, induced Fos in ipsilateral Vc; Fos expression was significantly greater in inflamed, stimulated animals. pERK was present in both neurons and astrocytes; pp38 was present in neurons and other non-neuronal, non-astrocytic cell types. CONCLUSIONS: This study provides the first demonstration that chronic inflammation of tooth pulp induces persistent bilateral activation of ERK and p38 within Vc, and that this activation is further increased by acute stimulation. This altered activity in intracellular signaling is likely to be linked to the sensitization that is seen in our animal model and in patients with pulpitis. Our data indicate that pERK and pp38 are more accurate markers of central change than Fos expression. In our model, localization of pERK and pp38 within specific cell types differs from that seen following acute stimulation. This may indicate specific roles for different cell types in the induction and maintenance of pulpitic and other types of pain.

M-channels modulate network excitatory activity induced by 4-aminopyridine in immature rat substantia gelatinosa in vitro.

There is strong evidence that M-currents modulate peripheral sensory afferent excitability and that altered M-current efficacy may underpin aspects of pain-induced nociceptor sensitization. Less clear is the role of the M-current in regulating central excitability within spinal dorsal horn nociceptive circuitry. In this study, an in vitro model of central hyperexcitability that uses the potassium channel blocker 4-aminopyridine (4-AP) to induce large amplitude population spikes and 4-12Hz oscillatory activity within rat spinal substantia gelatinosa (SG) has been used to determine the impact of pharmacological modulation of the M-current on central excitability. The M-current enhancers Retigabine (10 and 30µM) and Flupirtine (30µM) had a depressant effect on 4-AP-induced excitation in SG such that the frequency of large amplitude population spikes and the power of 4-12Hz oscillatory activity were both significantly reduced. In contrast, the M-current blockers XE911 (5µM) or Linopirdine (20µM) significantly potentiated 4-12Hz oscillatory activity as evidenced by significant increases in the parameters of power amplitude and power area but had no effect on large amplitude population spikes. These data indicate that pharmacological modulation of the M-current can influence excitability of nociceptive circuitry especially under conditions of central hyperexcitability, as may occur in chronic pain conditions. It is not clear whether these effects reflect a direct effect on interneurones localized to SG or indirectly via sensory afferent terminals. Nonetheless, these central actions should be taken into account alongside peripheral actions in terms of evaluating the potential therapeutic analgesic potency of novel M-current enhancers.

Localization of neurones expressing the gap junction protein Connexin45 within the adult spinal dorsal horn: a study using Cx45-eGFP reporter mice.

Connexin (Cx) proteins localized to neuronal and glial syncytia provide the ultrastructural components for intercellular communication via gap junctions. In this study, a Cx45 reporter mouse model in which the Cx45 coding sequence is substituted for enhanced green fluorescent protein (eGFP) was used to characterize Cx45 expressing neurones within adult mouse spinal cord. eGFP-immunoreactive (eGFP-IR) cells were localized at all rostro-caudal levels to laminae I-III of the dorsal horn (DH), areas associated with nociception. The neuronal rather than glial phenotype of these cells in DH was confirmed by co-localisation of eGFP-IR with the neuronal marker NeuN. Further immunohistochemical studies revealed that eGFP-IR interneurones co-express the calcium-binding protein calbindin, and to a lesser extent calretinin. In contrast, eGFP-IR profiles did not co-localize with either parvalbumin or GAD-67, both of which are linked to inhibitory interneurones. Staining with the primary afferent markers isolectin-B4 (IB4) and calcitonin gene-related peptide revealed that eGFP-IR somata within laminae I-III receive close appositions from the former, presumed non-peptidergic nociceptive afferents of peripheral origin. The presence of 5-HT terminals in close apposition to eGFP-IR interneuronal somata suggests modulation via descending pathways. These data demonstrate a highly localized expression of Cx45 in a population of interneurones within the mouse superficial dorsal horn. The implications of these data in the context of the putative role of Cx45 and gap junctions in spinal somatosensory processing and pain are discussed.

Kv3.1b and Kv3.3 channel subunit expression in murine spinal dorsal horn GABAergic interneurones.

GABAergic interneurones, including those within spinal dorsal horn, contain one of the two isoforms of the synthesizing enzyme glutamate decarboxylase (GAD), either GAD65 or GAD67. The physiological significance of these two GABAergic phenotypes is unknown but a more detailed anatomical and functional characterization may help resolve this issue. In this study, two transgenic Green Fluorescent Protein (GFP) knock-in murine lines, namely GAD65-GFP and GAD67-GFP (Δneo) mice, were used to profile expression of Shaw-related Kv3.1b and Kv3.3 K(+)-channel subunits in dorsal horn interneurones. Neuronal expression of these subunits confers specific biophysical characteristic referred to as 'fast-spiking'. Immuno-labelling for Kv3.1b or Kv3.3 revealed the presence of both of these subunits across the dorsal horn, most abundantly in laminae I-III. Co-localization studies in transgenic mice indicated that Kv3.1b but not Kv3.3 was associated with GAD65-GFP and GAD67-GFP immunopositive neurones. For comparison the distributions of Kv4.2 and Kv4.3 K(+)-channel subunits which are linked to an excitatory neuronal phenotype were characterized. No co-localization was found between GAD-GFP +ve neurones and Kv4.2 or Kv4.3. In functional studies to evaluate whether either GABAergic population is activated by noxious stimulation, hindpaw intradermal injection of capsaicin followed by c-fos quantification in dorsal horn revealed co-expression c-fos and GAD65-GFP (quantified as 20-30% of GFP +ve population). Co-expression was also detected for GAD67-GFP +ve neurones and capsaicin-induced c-fos but at a much reduced level of 4-5%. These data suggest that whilst both GAD65-GFP and GAD67-GFP +ve neurones express Kv3.1b and therefore may share certain biophysical traits, their responses to peripheral noxious stimulation are distinct.

Neuroprotective upregulation of endogenous α-synuclein precedes ubiquitination in cultured dopaminergic neurons.

α-Synuclein is the major protein component of Lewy bodies--the pathological hallmark of Parkinson's disease (PD) and Dementia with Lewy bodies (DLB). Its accumulation into intracellular aggregates is implicated in the process of Lewy body formation. However, its roles in both normal function, and disease, remain controversial. Using a novel model of chronic oxidative stress in cultured dopaminergic and cortical neurons, we report that endogenous α-synuclein is upregulated in response to low dose toxicity. This response is conserved between subpopulations of cortical and dopaminergic neurons, and confers relative resistance to apoptosis following secondary insult. Additional acute oxidative stress leads to intracellular accumulation of α-synuclein. These punctate deposits colocalize with ubiquitin, which is central to proteosome-mediated protein degeneration, and is the second major component of Lewy bodies. The current results imply that differential levels of α-synuclein expression may influence neuronal vulnerability in chronic neurodegenerative diseases. They further support a 'two hit' hypothesis for Lewy body formation, whereby mild stress causes a protective upregulation of α-synuclein. However, such increased levels of α-synuclein may drive its accumulation, following additional toxic insult. Finally, these results support a common mechanism for degeneration of dopaminergic and cortical neurons, affected in PD, and DLB, respectively.

Neuron-glia interactions underlie ALS-like axonal cytoskeletal pathology.

Amyotrophic lateral sclerosis (ALS) is a devastating disorder involving loss of movement due to degeneration of motor neurons. Studies suggest that in ALS axonal dysfunction precedes the death of motor neurons. Pathologically, ALS is characterized by neurofilamentous swellings (spheroids) within the axons of motor neurons. However, the causes of this axonopathy and possible resulting axonal dysfunction are not known. Using a novel model of cultured mouse motor neurons, we have determined that these neurons are susceptible to proximal axonopathy, which is related to the glial environment. This axonopathy showed remarkable similarity, both morphologically and neurochemically, to spheroids that develop over months in SOD1(G93A) transgenic mice. Focal ubiquitination, as well as perturbations of neurofilaments and microtubules, occurred in the axonal spheroid-like swellings in vitro, and visualization of mitochondrial dynamics demonstrated that axonopathy resulted in impaired axonal transport. These data provide strong evidence for the involvement of non-neuronal cells in axonal dysfunction in ALS. This cell culture model may be of benefit for the development of therapeutic interventions directed at axonal preservation.

An additive interaction between the NFkappaB and estrogen receptor signalling pathways in human endometrial epithelial cells.

BACKGROUND: Human embryo implantation is regulated by estradiol (E2), progesterone and locally produced mediators including interleukin-1beta (IL-1beta). Interactions between the estrogen receptor (ER) and NF kappa B (NFkappaB) signalling pathways have been reported in other systems but have not been detailed in human endometrium. METHODS AND RESULTS: Real-time PCR showed that mRNA for the p65 and p105 NFkappaB subunits is maximally expressed in endometrium from the putative implantation window. Both subunits are localized in the endometrial epithelium throughout the menstrual cycle. Reporter assays for estrogen response element (ERE) activity were used to examine functional interactions between ER and NFkappaB in telomerase immortalized endometrial epithelial cells (TERT-EEC). E2 and IL-1beta treatment of TERT-EECs enhances ERE activity by a NFkappaB and ER dependent mechanism; this effect could be mediated by ERalpha or ERbeta. E2 and IL-1beta also positively interact to increase endogenous gene expression of prostaglandin E synthase and c-myc. This is a gene-dependent action as there is no additive effect on cyclin D1 or progesterone receptor expression. CONCLUSION: In summary, we have established that NFkappaB signalling proteins are expressed in normal endometrium and report that IL-1beta can enhance the actions of E2 in a cell line derived from healthy endometrium. This mechanism may allow IL-1beta, possibly from the developing embryo, to modulate the function of the endometrial epithelium to promote successful implantation, for example by regulating prostaglandin production. Aberrations in the interaction between the ER and NFkappaB signalling pathways may have a negative impact on implantation contributing to pathologies such as early pregnancy loss and infertility.

Endometrial cysteine-rich secretory protein 3 is inhibited by human chorionic gonadotrophin, and is increased in the decidua of tubal ectopic pregnancy.

Ectopic pregnancy (EP) remains a considerable cause of morbidity and occasional mortality. Currently, there is no reliable test to differentiate ectopic from intrauterine gestation. We have previously used array technology to demonstrate that differences in gene expression in decidualized endometrium from women with ectopic and intrauterine gestations could be used to identify candidate diagnostic biomarkers for EP. The aim of this study was to further investigate the decidual gene with the highest fold increase in EP, cysteine-rich secretory protein-3 (CRISP-3). Decidualized endometrium from gestation-matched women undergoing surgical termination of pregnancy (n = 8), evacuation of uterus for miscarriage (n = 6) and surgery for EP (n = 11) was subjected to quantitative RT-PCR, morphological assessment, immunohistochemistry and western blot analysis. Sera were analysed for progesterone and human chorionic gonadotrophin (hCG) levels. Immortalized endometrial epithelial cells were cultured with physiological concentrations of hCG. CRISP-3 mRNA and protein expression were greater in endometrium from ectopic when compared with intrauterine pregnancies (P < 0.05). CRISP-3 protein was localized to epithelium and granulocytes of endometrium. CRISP-3 serum concentrations were not different in women with ectopic compared with intrauterine pregnancies. CRISP-3 expression in endometrium was not related to the degree of decidualization or to serum progesterone levels. Endometrial CRISP-3 expression was inversely proportional to serum hCG concentrations (P < 0.001). Stimulation of endometrial epithelial cells with hCG in vitro caused a reduction in CRISP-3 expression (P < 0.01). The measurement of CRISP-3 in endometrium could provide an additional tool in the diagnosis of failing early pregnancy of unknown location. The absence of a local reduction in expression of CRISP-3 in decidualized endometrium of women with EP may be due to reduced exposure to hCG due to the ectopic location of the trophoblast.

Altered secretory leukocyte protease inhibitor expression in the uterine decidua of tubal compared with intrauterine pregnancy.

BACKGROUND: The role of the innate immune system in tubal implantation remains undefined. This study compared expression of two key mediators of innate immunity, secretory leukocyte protease inhibitor (SLPI) and elafin, in the uterine decidua of women with intrauterine and tubal pregnancies. METHODS: Uterine decidua was collected from women (18-45 years) undergoing surgical termination of pregnancy (n = 7), surgical management of spontaneous abortion (n = 6) and tubal pregnancy (n = 10). Using quantitative RT-PCR and immunohistochemistry, mRNA and protein expression patterns of SLPI and elafin were compared. RESULTS: Relative SLPI mRNA expression was significantly higher in decidua of women with tubal pregnancy (12.37 +/- 2.66) compared with spontaneous abortion (5.09 +/- 2.22, P < 0.0185). There was no difference demonstrated in elafin mRNA expression. SLPI and elafin protein expression were demonstrated in the decidual leukocyte populations and epithelium. There was no obvious qualitative difference in levels of SLPI and elafin protein expression or their distribution in the uterine decidua of women with termination of pregnancy, spontaneous abortion or tubal pregnancy. CONCLUSIONS: Herein we demonstrate novel differences in gene expression of uterine decidua of tubal pregnancy compared with spontaneous abortion thereby contributing further to current knowledge of mechanisms involved in extrauterine implantation. The altered expression of SLPI may be a consequence of, or predispose to, tubal pregnancy.

Endometrial inhibin/activin beta-B subunit expression is related to decidualization and is reduced in tubal ectopic pregnancy.

CONTEXT: Ectopic pregnancy is common but remains difficult to diagnose accurately. There is no serum test to differentiate ectopic from intrauterine gestation. OBJECTIVE: Our objective was to investigate differential gene expression in decidualized endometrium of ectopic pregnancy. DESIGN: Tissue and serum analysis informed by microarray study was performed. SETTING: The study was performed at a large United Kingdom teaching hospital. PATIENTS OR OTHER PARTICIPANTS: Women undergoing surgical termination of pregnancy (n = 8), evacuation of uterus for miscarriage (n = 6), and surgery for tubal ectopic pregnancy (n = 11) were included in the study. Endometrium was collected from normally cycling women undergoing hysterectomy. INTERVENTIONS: Decidualized endometrium was subjected to microarray analysis, morphological assessment, and immunohistochemistry. Endometrial stromal fibroblasts were cultured in the presence of decidualizing stimuli. MAIN OUTCOME MEASURES: Differential expression of potentially secreted molecules was calculated. RESULTS: Inhibin/activin beta-B expression was lower in decidualized endometrium from ectopic pregnancies when compared with that of ongoing pregnancies (P < 0.01) or miscarriages (P < 0.01). The localization of the beta-B subunit was more marked in decidualized than nondecidualized stroma. Decidualization of stromal fibroblasts in vitro was associated with increased beta-B expression (P < 0.05). Endometrial stroma of ectopic pregnancies was less decidualized morphologically (P < 0.05), with lower prolactin (P < 0.01) and IGF binding protein-1 (P < 0.005) expression. Serum activin B was lower in ectopic pregnancies (P < 0.005) than in intrauterine pregnancies, whereas there was no difference in progesterone concentrations. CONCLUSIONS: Despite similar concentrations of progesterone, the endometrium of ectopic pregnancies is less decidualized than intrauterine pregnancies. Expression of the beta-B subunit is related to decidualization and can be detected in the circulation as activin B. Serum activin B concentrations are lower in ectopic pregnancy.

Excitotoxicity mediated by non-NMDA receptors causes distal axonopathy in long-term cultured spinal motor neurons.

Excitotoxicity has been implicated as a potential cause of neuronal degeneration in amyotrophic lateral sclerosis (ALS). It has not been clear how excitotoxic injury leads to the hallmark pathological changes of ALS, such as the abnormal accumulation of filamentous proteins in axons. We have investigated the effects of overactivation of excitatory receptors in rodent neurons maintained in long-term culture. Excitotoxicity, mediated principally via non-N-methyl-D-aspartate (NMDA) receptors, caused axonal swelling and accumulation of cytoskeletal proteins in the distal segments of the axons of cultured spinal, but not cortical, neurons. Axonopathy only occurred in spinal neurons maintained for 3 weeks in vitro, indicating that susceptibility to axonal pathology may be related to relative maturity of the neuron. Excitotoxic axonopathy was associated with the aberrant colocalization of phosphorylated and dephosphorylated neurofilament proteins, indicating that disruption to the regulation of phosphorylation of neurofilaments may lead to their abnormal accumulation. These data provide a strong link between excitotoxicity and the selective pattern of axonopathy of lower motor neurons that underlies neuronal dysfunction in ALS.

Innate immune defences in the human uterus during pregnancy.

The prevention of uterine infection is critical to appropriate fetal development and term delivery. The innate immune system is one component of the uterine environment and has a role in prevention of uterine infection. Natural antimicrobials are innate immune molecules with anti-bacterial, anti-viral and anti-fungal activity. We discuss two groups of natural antimicrobials in relation to pregnancy: (i) the defensins; and (ii) the whey acidic protein motif containing proteins, secretory leukocyte protease inhibitor (SLPI) and elafin. Human beta-defensins (HBD) 1-3 are expressed by placental and chorion trophoblast, amnion epithelium and decidua in term and preterm pregnancy. Elafin shows a similar pattern of localisation while SLPI is produced only by amnion epithelium and decidua. Evidence suggests that there is aberrant production of some natural antimicrobials in pathologic conditions of pregnancy. In preterm premature rupture of membranes (PPROM) levels of SLPI and elafin are reduced in amniotic fluid and fetal membranes, respectively. Elafin and HBD3 increase in chorioamnionitis and levels of the alpha-defensins, HNP1-3, increase in maternal plasma and amniotic fluid in women affected by microbial invasion of the uterus. In vitro culture studies have suggested a mechanism for increased production of natural antimicrobials in chorioamnionitis. Elafin, SLPI, HBD2 and 3 are all upregulated by inflammatory molecules in cells derived from gestational tissues. In summary, production of natural antimicrobials at key sites within the pregnant uterus suggests an important role in prevention of uterine infection during pregnancy and labour. Aberrant production of these molecules in PPROM and chorioamnionitis suggests that they also have a role in pathologic conditions. In particular, upregulation of these molecules by inflammatory molecules present in chorioamnionitis will ensure a robust response to infection.

Differential activation of protein kinases in the dorsal horn in vitro of normal and inflamed rats by group I metabotropic glutamate receptor subtypes.

Group I metabotropic glutamate receptors (mGluRs) contribute to spinal sensitization and synaptic plasticity but the underlying mechanisms are unknown. Here, group I mGluR modulation of evoked monosynaptic excitatory postsynaptic currents (EPSCs) in substantia gelatinosa (SG) neurones in vitro was investigated in juvenile rats. In addition, the role of group I mGluRs in dorsal horn neuronal Fos expression was determined in tetrodotoxin (TTX)-treated in vitro spinal cords of naïve rats and those with Complete Freund's Adjuvant (CFA) peripheral inflammation. In the majority of SG neurones, (S)-3,5-dihydroxyphenylglycine (DHPG) reduced EPSCs and this effect was inhibited by the mGluR(5) antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP). Data for paired-pulse and spontaneous miniature excitatory postsynaptic currents (mEPSCs) suggest mGluR(5) acts presynaptically to reduce transmitter release. DHPG-induced reduction of EPSC amplitude operated via PKC, but not ERK, signalling cascade. In the dorsal horn of naïve but not CFA rats, DHPG increased Fos expression and this was reduced by MPEP and both PKC and ERK inhibitors. In the CFA group, basal Fos expression was reduced by MPEP and the kinase inhibitors. These data infer a role for mGluR(5) in acute modulation of nociceptive synaptic efficacy within the dorsal horn and postsynaptic activation of transcription factors such as Fos that are implicated in activity-dependent neuroplastic adaptation. These actions are achieved by differential activation of PKC- and ERK-dependent transduction pathways.

Expression of natural antimicrobials by human placenta and fetal membranes.

Preterm birth associated with infection is a major clinical problem. We hypothesized that this condition is associated with altered expression of natural antimicrobial molecules (beta-defensins (HBD), elafin). Therefore, we examined expression of these molecules and their regulation by proinflammatory cytokines in placentae and fetal membranes from term pregnancy. HBD1-3 and elafin were localized by immunohistochemistry in fetal membranes and placenta. Real-time quantitative PCR was used to examine mRNA expression in primary trophoblast cells treated with inflammatory molecules. HBD1-3 and elafin were immunolocalized to placental and chorion trophoblast layers of fetal membranes and placenta. Immunoreactivity was also observed in amnion epithelium and decidua. No differences were noted between samples from women who were not in labour compared to those in active labour. In in vitro cultures of primary trophoblast cells, HBD2 and elafin mRNA expression was upregulated by the proinflammatory cytokine, IL-1beta. These results suggest that the chorion and placental trophoblast layers may be key barriers to the progression of infection in the pregnant uterus. Natural antimicrobial expression may be altered in response to inflammatory mediator expression associated with the onset of labour and/or uterine infection, providing increased protection when the uterus may be particularly susceptible to infection.

Localization of glutamate receptors in developing cortical neurons in culture and relationship to susceptibility to excitotoxicity.

Overactivation of glutamate receptors leading to excitotoxicity has been implicated in the neurodegenerative alterations of a range of central nervous system (CNS) disorders. We have investigated the cell-type-specific changes in glutamate receptor localization in developing cortical neurons in culture, as well as the relationship between glutamate receptor subunit distribution with synapse formation and susceptibility to excitotoxicity. Glutamate receptor subunit clustering was present prior to the formation of synapses. However, different receptor types showed distinctive temporal patterns of subunit clustering, localization to spines, and apposition to presynaptic terminals. N-methyl-D-aspartate (NMDA) receptor subunit immunolabelling was present in puncta along dendrites prior to the formation of synapses, with relatively little localization to spines. Vulnerability to NMDA receptor-mediated excitotoxicity occurred before receptor subunits became localized in apposition to presynaptic terminals. Clustering of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors occurred concurrently with development of vulnerability to excitotoxicity and was related to localization of AMPA receptors at synapses and in spines. Different AMPA receptor subunits demonstrated cell-type-specific localization as well as distribution to spines, dendrites, and extrasynaptic subunit clusters. A subclass of neurons demonstrated substantial perineuronal synaptic innervation, and these neurons expressed relatively high levels of GluR1 and/or GluR4 at receptor puncta, indicating the presence of calcium-permeable AMPA receptors and suggesting alternative synaptic signalling mechanisms and vulnerability to excitotoxicity. These data demonstrate the relationship between glutamate receptor subunit expression and localization with synaptogenesis and development of neuronal susceptibility to excitotoxicity. These data also suggest that excitotoxicity can be mediated through extrasynaptic receptor subunit complexes along dendrites.