Lactobacilli displacement and Candida albicans inhibition on initial adhesion assays: a probiotic analysis.

OBJECTIVE: This study evaluates the probiotic activity of three vaginal Lactobacillus gasseri (H59.2, IMAUFB014, and JCM1131) and one non-vaginal L. plantarum ATCC14917 against three Candida albicans (ATCC10231, candidiasis, and healthy vaginal microbiota). Displacement of lactobacilli and adhesion inhibition of C. albicans were evaluated on an abiotic surface through adhesion assays with different experimental settings (ES) through low (1.0E + 03 CFU/ml) and high (1.00E + 09 CFU/ml) levels of colonization. ES simulated dysbiosis (ES1 and ES4), candidiasis (ES2), and healthy vaginal microbiota (ES3). RESULTS: At ES2 and ES3, L. gasseri H59.2 showed discrepant inhibition values among C. albicans isolates (ES2: P = 0.008, ES3: P = 0.030; two-way ANOVA). L. plantarum was only displaced by 23%, 31%, 54%, and 94% against low and high levels of C. albicans ATCC10231. L. plantarum was less displaced, when compared to L. gasseri strains (ES1: 61-84%, ES2: 82-96%, ES3: 83-95%, and ES4: 73-97%), showing multiple statistical differences (ES1: P = < 0.001, ES2: P = 0.003, and ES3: P = < 0.001; two-way ANOVA). L. plantarum also showed a superior inhibition of C. albicans ATCC10231 in ES1 (81%) and ES2 (58%) when compared to L. gasseri strains (ES1: 27-73%, P < 0.001; and ES2:1-49%, P < 0.001; two-way ANOVA).

Acid sensing ion channel (ASIC) inhibitors exhibit anxiolytic-like activity in preclinical pharmacological models.

RATIONALE: Acid sensing ion channels (ASICs) are proton-gated ion channels located in the central and peripheral nervous systems. Of particular interest is ASIC1a, which is located in areas associated with fear and anxiety behaviors. Recent reports suggest a role for ASIC1a in preclinical models of fear conditioning and anxiety. OBJECTIVES: The present experiments evaluated various ASIC inhibitors in preclinical models of autonomic and behavioral parameters of anxiety. In addition, neurochemical studies evaluated the effects of an ASIC inhibitor (A-317567) on neurotransmitter levels in the amygdala. RESULTS: In electrophysiological studies using hippocampal primary neuronal cultures, three ASIC inhibitors (PcTX-1, A-317567, and amiloride) produced concentration-dependent inhibition of acid-evoked currents. In the stress-induced hyperthermia model, acute administration of psalmotoxin 1 (PcTX-1; 10-56 ng, i.c.v.), A-317567 (0.1-1.0 mg/kg, i.p.), and amiloride (10-100 mg/kg, i.p.) prevented stress-induced elevations in core body temperature. In the four-plate test, acute treatment with PcTX-1 (10-56 ng, i.c.v.) and A-317567 (0.01-0.1 mg/kg, i.p.), but not amiloride (3-100 mg/kg, i.p.), produced dose-dependent and significant increases in the number of punished crossings relative to vehicle-treated animals. Additionally, PcTX-1 (56-178 ng, i.c.v.), A-317567 (0.1-10 mg/kg, i.p.), and amiloride (10-100 mg/kg, i.p.) lacked significant anxiolytic-like activity in the elevated zero maze. In neurochemical studies, an infusion of A-317567 (100 microM) into the amygdala significantly elevated the extracellular levels of GABA, but not glutamate, in this brain region. CONCLUSIONS: These findings demonstrate that ASIC inhibition produces anxiolytic-like effects in some behavioral models and indicate a potential role for GABAergic mechanisms to underlie these anxiolytic-like effects.

Amiloride is neuroprotective in an MPTP model of Parkinson's disease.

The diuretic amiloride has recently proven neuroprotective in models of cerebral ischemia, a property attributable to the drug's inhibition of central acid-sensing ion channels (ASICs). Given that Parkinson's disease (PD), like ischemia, is associated with cerebral lactic acidosis, we tested amiloride in the MPTP-treated mouse, a model of PD also manifesting lactic acidosis. Amiloride was found to protect substantia nigra (SNc) neurons from MPTP-induced degeneration, as determined by attenuated reductions in striatal tyrosine hydroxylase (TH) and dopamine transporter (DAT) immunohistochemistry, as well as smaller declines in striatal DAT radioligand binding and dopamine levels. More significantly, amiloride also preserved dopaminergic cell bodies in the SNc. Administration of psalmotoxin venom (PcTX), an ASIC1a blocker, resulted in a much more modest effect, attenuating only the deficits in striatal DAT binding and dopamine. These findings represent the first experimental evidence of a potential role for ASICs in the pathogenesis of Parkinson's disease.

Enhanced clearance of Abeta in brain by sustaining the plasmin proteolysis cascade.

The amyloid hypothesis states that a variety of neurotoxic beta-amyloid (Abeta) species contribute to the pathogenesis of Alzheimer's disease. Accordingly, a key determinant of disease onset and progression is the appropriate balance between Abeta production and clearance. Enzymes responsible for the degradation of Abeta are not well understood, and, thus far, it has not been possible to enhance Abeta catabolism by pharmacological manipulation. We provide evidence that Abeta catabolism is increased after inhibition of plasminogen activator inhibitor-1 (PAI-1) and may constitute a viable therapeutic approach for lowering brain Abeta levels. PAI-1 inhibits the activity of tissue plasminogen activator (tPA), an enzyme that cleaves plasminogen to generate plasmin, a protease that degrades Abeta oligomers and monomers. Because tPA, plasminogen and PAI-1 are expressed in the brain, we tested the hypothesis that inhibitors of PAI-1 will enhance the proteolytic clearance of brain Abeta. Our data demonstrate that PAI-1 inhibitors augment the activity of tPA and plasmin in hippocampus, significantly lower plasma and brain Abeta levels, restore long-term potentiation deficits in hippocampal slices from transgenic Abeta-producing mice, and reverse cognitive deficits in these mice.

High-throughput electrophysiology: an emerging paradigm for ion-channel screening and physiology.

Ion channels represent highly attractive targets for drug discovery and are implicated in a diverse range of disorders, in particular in the central nervous and cardiovascular systems. Moreover, assessment of cardiac ion-channel activity of new chemical entities is now an integral component of drug discovery programmes to assess potential for cardiovascular side effects. Despite their attractiveness as drug discovery targets ion channels remain an under-exploited target class, which is in large part due to the labour-intensive and low-throughput nature of patch-clamp electrophysiology. This Review provides an update on the current state-of-the-art for the various automated electrophysiology platforms that are now available and critically evaluates their impact in terms of ion-channel screening, lead optimization and the assessment of cardiac ion-channel safety liability.

Activation of estrogen receptor-beta regulates hippocampal synaptic plasticity and improves memory.

Estrogens have long been implicated in influencing cognitive processes, yet the molecular mechanisms underlying these effects and the roles of the estrogen receptors alpha (ERalpha) and beta (ERbeta) remain unclear. Using pharmacological, biochemical and behavioral techniques, we demonstrate that the effects of estrogen on hippocampal synaptic plasticity and memory are mediated through ERbeta. Selective ERbeta agonists increased key synaptic proteins in vivo, including PSD-95, synaptophysin and the AMPA-receptor subunit GluR1. These effects were absent in ERbeta knockout mice. In hippocampal slices, ERbeta activation enhanced long-term potentiation, an effect that was absent in slices from ERbeta knockout mice. ERbeta activation induced morphological changes in hippocampal neurons in vivo, including increased dendritic branching and increased density of mushroom-type spines. An ERbeta agonist, but not an ERalpha agonist, also improved performance in hippocampus-dependent memory tasks. Our data suggest that activation of ERbeta can regulate hippocampal synaptic plasticity and improve hippocampus-dependent cognition.

Early-onset behavioral and synaptic deficits in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder for which numerous mouse models have been generated. In both AD patients and mouse models, there is increasing evidence that neuronal dysfunction occurs before the accumulation of beta-amyloid (Abeta)-containing plaques and neurodegeneration. Characterization of the timing and nature of preplaque dysfunction is important for understanding the progression of this disease and to identify pathways and molecular targets for therapeutic intervention. Hence, we have examined the progression of dysfunction at the morphological, functional, and behavioral levels in the Tg2576 mouse model of AD. Our data show that decreased dendritic spine density, impaired long-term potentiation (LTP), and behavioral deficits occurred months before plaque deposition, which was first detectable at 18 months of age. We detected a decrease in spine density in the outer molecular layer of the dentate gyrus (DG) beginning as early as 4 months of age. Furthermore, by 5 months, there was a decline in LTP in the DG after perforant path stimulation and impairment in contextual fear conditioning. Moreover, an increase in the Abeta42/Abeta40 ratio was first observed at these early ages. However, total amyloid levels did not significantly increase until approximately 18 months of age, at which time significant increases in reactive astrocytes and microglia could be observed. Overall, these data show that the perforant path input from the entorhinal cortex to the DG is compromised both structurally and functionally, and this pathology is manifested in memory defects long before significant plaque deposition.

Beta estrogen receptor knockout (BERKO) mice present attenuated hippocampal CA1 long-term potentiation and related memory deficits in contextual fear conditioning.

Estrogen has marked effects on hippocampal synaptic plasticity. We demonstrate that male and female 3-month-old beta estrogen receptor knockout (BERKO) mice show profound memory impairment in a hippocampus-mediated fear-conditioning paradigm. Subsequently, hippocampal slices prepared from behaviorally naive female BERKO mice were examined electrophysiologically. These were found to have robust synaptic deficits, compared to slices from age-matched wild type controls, both in terms of their input-output curves and their expression of long-term-potentiation in area CA1. This report provides the first concrete evidence of significant hippocampal synaptic plasticity and memory deficits in the BERKO mouse.

Pharmacological characterization of antiepileptic drugs and experimental analgesics on low magnesium-induced hyperexcitability in rat hippocampal slices.

Perfusion of acute hippocampal slices with stimulatory buffers has long been known to induce rhythmic, large amplitude, synchronized spontaneous neuronal bursting in areas CA1 and CA3. The characteristics of this model of neuronal hyperexcitability were investigated in this study, particularly with respect to the activity of antiepileptic drugs and compounds representing novel mechanisms of analgesic action. Toward that end, low Mg(2+)/high K(+)-induced spontaneous activity was quantified by a virtual instrument designed for the digitization and analysis of bursting activity. Uninterrupted streams of extracellular field potentials were digitized and analyzed in 10-s sweeps, yielding four quantified parameters of neuronal hyperexcitability. Following characterization of the temporal stability of low Mg(2+)/high K(+)-induced hyperexcitability, compounds representing a diversity of functional mechanisms were tested for their effectiveness in reversing this activity. Of the four antiepileptic drugs tested in this model, only phenytoin proved ineffective, while valproate, gabapentin and carbamazepine varied in their potencies, with only the latter drug proving to be completely efficacious. In addition, three investigational compounds having analgesic potential were examined: ZD-7288, a blocker of HCN channels; EAA-090, an NMDA antagonist; and WAY-132983, a muscarinic agonist. Each of these compounds showed strong efficacy by completely blocking spontaneous bursting activity, along with potency greater than that of the antiepileptic drugs. These data indicate that pharmacological agents with varying mechanisms of action are able to block low Mg(2+)/high K(+)-induced hyperexcitability, and thus this model may represent a useful tool for identifying novel agents and mechanisms involved in epilepsy and neuropathic pain.