automated-quantitative-cognitivebehavioral-screening-mice-for

Automated, Quantitative Cognitive/Behavioral Screening of Mice: For Genetics, Pharmacology, Animal Cognition and Undergraduate Instruction

We describe a high-throughput, high-volume, fully automated, live-in 24/7 behavioral testing system for assessing the effects of genetic and ...

The cellular effects of biodiesel emissions particulate matter (BDEP) and petroleum diesel emissions particulate matter (PDEP) were compared using a human airway cell line, A549. At concentrations of 25 microg/ml, diesel particulate matter induced the formation of multinucleate cells. In cells treated with a mixture of 80% PDEP:20% BDEP, 52% of cells were multinucleate cells compared with only 16% of cells treated with 20% PDEP:80% BDEP with a background multinucleate rate of 7%. These results demonstrate a causal relation between the formation of multinucleate cells and exposure to exhaust particulate matter, in particular diesel exhaust. Exposure of A549 cells to PDEP induced apoptosis, seen by active caspase-3 expression and the presence of cleaved pancytokeratin. PDEP exhaust was a much stronger inducer of cellular death through apoptosis than BDEP. There was an eightfold increase in the expression of SLC30A3 (zinc transporter-3 or ZnT3) in cells exposed to 80% PDEP:20% BDEP compared to untreated cells. The increase in ZnT3 expression seen in apoptotic cells following PDEP suggests a role for this zinc transporter in the apoptotic pathway, possibly through controlling zinc fluxes. As exposure to diesel exhaust particles is associated with asthma and apoptosis in airway cells, diesel exhaust particles may directly contribute to asthma by inducing epithelial cell death through apoptotic pathway.

Diesel exhaust particulate matter induces multinucleate cells and zinc transporter-dependent apoptosis in human airway cells.

A role for the copper transporter, ATP7B, in secretion of copper from the human breast into milk has previously not been reported, although it is known that the murine ortholog of ATP7B facilitates copper secretion in the mouse mammary gland. We show here that ATP7B is expressed in luminal epithelial cells in both the resting and lactating human breast, where it has a perinuclear localization in resting epithelial cells and a diffuse location in lactating tissue. ATP7B protein was present in a different subset of vesicles from those containing milk proteins and did not overlap with Menkes ATPase, ATP-7A, except in the perinuclear region of cells. In the cultured human mammary line, PMC42-LA, treatment with lactational hormones induced a redistribution of ATP7B from a perinuclear region to a region adjacent, but not coincident with, the apical plasma membrane. Trafficking of ATP7B was copper dependent, suggesting that the hormone-induced redistribution of ATP7A was mediated through an increase in intracellular copper. Radioactive copper ((64)Cu) studies using polarized PMC42-LA cells that overexpressed mAtp7B protein showed that this transporter facilitates copper efflux from the apical surface of the cells. In summary, our results are consistent with an important function of ATP7B in the secretion of copper from the human mammary gland.

ATP7B expression in human breast epithelial cells is mediated by lactational hormones.

Both copper transporting ATPases, ATP7A and ATP7B, are expressed in mammary epithelial cells but their role in copper delivery to milk has not been clarified. We investigated the role of ATP7A in delivery of copper to milk using transgenic mice that over-express human ATP7A. In mammary gland of transgenic mice, human ATP7A protein was 10- to 20-fold higher than in control mice, and was localized to the basolateral membrane of mammary epithelial cells in lactating mice. The copper concentration in the mammary gland of transgenic dams and stomach contents of transgenic pups was significantly reduced compared to non-transgenic mice. The mRNA levels of endogenous Atp7a, Atp7b, and Ctr1 copper transporters in the mammary gland were not altered by the expression of the ATP7A transgene, and the protein levels of Atp7b and ceruloplasmin were similar in transgenic and non-transgenic mice. These data suggest that ATP7A plays a role in removing excess copper from the mammary epithelial cells rather than supplying copper to milk.

Copper transport during lactation in transgenic mice expressing the human ATP7A protein.

Zinc homeostasis was investigated in Nostoc punctiforme. Cell tolerance to Zn(2+) over 14 days showed that ZnCl(2) levels above 22 microM significantly reduced cell viability. After 3 days in 22 microM ZnCl(2), ca. 12% of the Zn(2+) was in an EDTA-resistant component, suggesting an intracellular localization. Zinquin fluorescence was detected within cells exposed to concentrations up to 37 microM relative to 0 microM treatment. Radiolabeled (65)Zn showed Zn(2+) uptake increased over a 3-day period, while efflux occurred more rapidly within a 3-h time period. Four putative genes involved in Zn(2+) uptake and efflux in N. punctiforme were identified: (i) the predicted Co/Zn/Cd cation transporter, putative CDF; (ii) the predicted divalent heavy-metal cation transporter, putative Zip; (iii) the ATPase component and Fe/Zn uptake regulation protein, putative Fur; and (iv) an ABC-type Mn/Zn transport system, putative zinc ZnuC, ZnuABC system component. Quantitative real-time PCR indicated the responsiveness of all four genes to 22 microM ZnCl(2) within 3 h, followed by a reduction to below basal levels after 24 h by putative ZIP, ZnuC, and Fur and a reduction to below basal level after 72 h by putative CDF efflux gene. These results demonstrate differential regulation of zinc transporters over time, indicating a role for them in zinc homeostasis in N. punctiforme.

Bioinformatic and expression analyses of genes mediating zinc homeostasis in Nostoc punctiforme.

Human and mouse subjects tried to anticipate at which of 2 locations a reward would appear. On a randomly scheduled fraction of the trials, it appeared with a short latency at one location; on the complementary fraction, it appeared after a longer latency at the other location. Subjects of both species accurately assessed the exogenous uncertainty (the probability of a short versus a long trial) and the endogenous uncertainty (from the scalar variability in their estimates of an elapsed duration) to compute the optimal target latency for a switch from the short- to the long-latency location. The optimal latency was arrived at so rapidly that there was no reliably discernible improvement over trials. Under these nonverbal conditions, humans and mice accurately assess risks and behave nearly optimally. That this capacity is well-developed in the mouse opens up the possibility of a genetic approach to the neurobiological mechanisms underlying risk assessment.

Risk assessment in man and mouse.

Docosahexaenoic acid (DHA) is the major polyunsaturated fatty acid in neuronal cell membranes. We hypothesize that DHA induces a decrease in neuronal cell death through reduced ZnT3 expression and zinc uptake. Exposure of M17 cells to DHA-deficient medium increased the levels of active caspase-3, relative to levels in DHA-replete cells, confirming the adverse effects of DHA deficiency in promoting neuronal cell death. In DHA-treated M17 cells, zinc uptake was 65% less and ZnT3 mRNA and protein levels were reduced in comparison with DHA-depleted cells. We propose that the neuroprotective function of DHA is exerted through a reduction in cellular zinc levels that in turn inhibits apoptosis.

The omega-3 fatty acid, DHA, decreases neuronal cell death in association with altered zinc transport.

Docosahexaens&auml;ure (DHA) ist die wichtige mehrfach unges&auml;ttigte Fetts&auml;ure in neuronalen Zellmembranen. Wir vermuten, dass DHA einen R&uuml;ckgang der neuronalen Zelltod durch reduzierte ZnT3 Ausdruck und Zink-Aufnahme induziert. Aussetzung M17 Zellen zum Medium der DHA-Mangel erh&ouml;ht die Ebenen der aktive Caspase-3, relativ Ebenen in DHA-vollgestopft Zellen, best&auml;tigen die negativen Auswirkungen der DHA-Mangel bei der F&ouml;rderung der neuronalen Zelltod. M17 DHA-behandelten Zellen Zink-Aufnahme war 65 % weniger und ZnT3 mRNA und Protein im Vergleich zu DHA-abgereichertem Zellen reduziert wurden. Wir schlagen die neuroprotektive Funktion von DHA durch zellul&auml;re Zink abbauen ausge&uuml;bt wird, die wiederum hemmt die Apoptose.

Die Omega-3-Fetts&auml;ure DHA, vermindert neuronalen Zelltod in Verbindung mit ver&auml;nderten Zink-Transport.

The goal was to determine whether a signal (e.g., a click) at food availability affects timing behavior in rats. Twenty-four rats were trained on an appetitive lever-press procedure that varied on two dimensions: shape of the interreinforcer distribution (i.e., fixed-interval 60s or random-interval 60s) and number of signals (i.e., the presence or absence of a click at the time of reinforcer availability). The rats were randomly partitioned into one of four groups (each group had six rats): Fixed, Signaled-Fixed, Random, and Signaled-Random. The shape of the interreinforcer distribution affected the response pattern; the presence of the click affected response rate. These results provide support for a simultaneous temporal processing account of behavior.

A simultaneous temporal processing account of response rate.

Shifts in the psychometric function in the Free Operant Psychophysical Procedure (FOPP) have traditionally been explained by suggesting the pacemaker rate is proportional to the reinforcement rate (Behavioral Theory of Timing; BeT), or by direct associative competition between the two responses (Learning to Time; LeT). The application of these models assumes that the stimulus onset is the relevant time marker. Head-entries were reinforced in the second half of a stimulus (s1) and in the first half of a different stimulus (s2). During extinction, response times shifted earlier in s2 only, contrary to BeT. Competition between responses is an unlikely cause of the shift, contrary to LeT. We found a single-cycle correlation between the last food delivery and the time the rat stopped responding on s2. This correlation was also present in FOPP data. These results are consistent with the interpretation that the reinforcer, more than the stimulus onset, acted as the relevant time marker on this task.

The importance of the reinforcer as a time marker.

To gain insight into the reproductive toxicity of sublethal cadmium on marine polychaetes, Perinereis nuntia sandworms were exposed to cadmium via artificially contaminated seawater. Cadmium influence on sexual maturation, egg laying, fertilization, zygote hatching and vitellogenin expression levels were analyzed. Results indicated that 23.05 and 563.87 microg L(-1) cadmium significantly delayed sexual maturation. Fertilization rate was significantly inhibited by 563.87 microg L(-1) cadmium while lower concentrations showed no significant effect. Zygote hatching was significantly inhibited by cadmium concentrations greater than 1.12 microg L(-1). We identified a vitellogenin gene sequence in P. nuntia and found that mRNA transcription was significantly upregulated by cadmium. These results indicate that sublethal cadmium levels cause dose-dependent reproductive toxicity on P. nuntia by inhibiting sexual maturation, fertilization and zygote hatching, and the increased expression of vitellogenin suggesting cadmium has strong feminization effects on polychaetes.

Reproductive toxic effects of sublethal cadmium on the marine polychaete Perinereis nuntia.

Um Einblick in die Reproduktionstoxizit&auml;t subletale Cadmium auf marine Polychaetes, waren Perinereis Nuntia Wattw&uuml;rmer Kadmium &uuml;ber k&uuml;nstlich kontaminierten Meerwasser ausgesetzt. Kadmium Einfluss auf sexuelle Reifung, egg, Verlegung, Befruchtung, Zygote schl&uuml;pfen und Vitellogenine Stufen wurden analysiert. Ergebnisse zeigten, dass 23.05 und 563.87 Microg L(-1) Kadmium deutlich sexuelle Reifung verz&ouml;gert. Befruchtung Satz wurde deutlich durch 563.87 Microg L(-1) Cadmium gehemmt, w&auml;hrend niedrigere Konzentrationen keine signifikanten Auswirkungen zeigte. Zygote Schraffur wurde wesentlich durch Cadmium-Konzentrationen gr&ouml;&szlig;er 1,12 Microg L(-1) gehemmt. Wir eine Gensequenz Vitellogenine in p Nuntia identifiziert und fand, da&szlig; mRNA-Transkription deutlich herraufreguliert durch Cadmium. Diese Ergebnisse zeigen, dass subletale Cadmiumgehalt dosisabh&auml;ngige Reproduktionstoxizit&auml;t auf s. Nuntia verursachen, durch die Hemmung der sexuellen Reifung, D&uuml;ngung und Zygote schl&uuml;pfen und die erh&ouml;hte Expression von Vitellogenine vorschlagen Kadmium starke Feminisierung Auswirkungen auf Polychaetes hat.

Reproduktive toxische Wirkungen von subletale Cadmium auf die marine Vielborster Perinereis Nuntia.

To gain insight into the antibiotic pollution in the Jiulong River estuary and the pollutant sources, we analyzed the concentration of 22 widely-used antibiotics in water samples collected from the river and estuary, 17 and 18 sampling sites, respectively. Contamination with sulfonamides, quinolones and chloramphenicols was frequently detected and the distribution pattern of antibiotics suggested that most of the pollutants are from the Jiulong River, especially from the downstream watersheds. To reveal the ecological effects, we isolated 35 bacterial strains from the estuary and analyzed their antibiotic resistance to the eight most frequently detected antibiotics. The bacteria were subsequently classified into seven different genera by 16SrDNA sequencing. Up to 97.1% of the bacteria showed resistance and 70.6% of strains showed multi-resistance to these antibiotics, especially to sulfonamides. This study demonstrated a pattern of antibiotic contamination in the Jiulong River and its estuary and illustrated high bacterial antibiotic resistance which was significantly correlated with the average antibiotics concentrations and detected frequencies in the estuary.

Antibiotics pollution in Jiulong River estuary: source, distribution and bacterial resistance.

Time is an essential feature of most decisions, because the reward earned from decisions frequently depends on the temporal statistics of the environment (e.g., on whether decisions must be made under deadlines). Accordingly, evolution appears to have favored a mechanism that predicts intervals in the seconds to minutes range with high accuracy on average, but significant variability from trial to trial. Importantly, the subjective sense of time that results is sufficiently imprecise that maximizing rewards in decision-making can require substantial behavioral adjustments (e.g., accumulating less evidence for a decision in order to beat a deadline). Reward maximization in many daily decisions therefore requires optimal temporal risk assessment. Here, we review the temporal decision-making literature, conduct secondary analyses of relevant published datasets, and analyze the results of a new experiment. The paper is organized in three parts. In the first part, we review literature and analyze existing data suggesting that animals take account of their inherent behavioral variability (their "endogenous timing uncertainty") in temporal decision-making. In the second part, we review literature that quantitatively demonstrates nearly optimal temporal risk assessment with sub-second and supra-second intervals using perceptual tasks (with humans and mice) and motor timing tasks (with humans). We supplement this section with original research that tested human and rat performance on a task that requires finding the optimal balance between two time-dependent quantities for reward maximization. This optimal balance in turn depends on the level of timing uncertainty. Corroborating the reviewed literature, humans and rats exhibited nearly optimal temporal risk assessment in this task. In the third section, we discuss the role of timing uncertainty in reward maximization in two-choice perceptual decision-making tasks and review literature that implicates timing uncertainty as an important factor in performance quality. Together, these studies strongly support the hypothesis that animals take normative account of their endogenous timing uncertainty. By incorporating the psychophysics of interval timing into the study of reward maximization, our approach bridges empirical and theoretical gaps between the interval timing and decision-making literatures.

Optimal temporal risk assessment.

Copper-based compounds have been used as agricultural fungicides for many years. Their use in Australia is escalating with increase in the scale of planting and associated pest problems. The objective of this study was to identify viticulture activities associated with high exposure to foliage sprays. It would be determined if occupational exposure of vineyard workers to copper-based sprays was associated with raised body copper levels through analysis of saliva and buccal cells.

Copper levels in buccal cells of vineyard workers engaged in various activities.

Adequate amounts of copper in milk are critical for normal neonatal development, however the mechanisms regulating copper supply to milk have not been clearly defined. PMC42-LA cell cultures representative of resting, lactating and suckled mammary epithelia were used to investigate the regulation of the copper uptake protein, CTR1. Both the degree of mammary epithelial differentiation (functionality) and extracellular copper concentration greatly impacted upon CTR1 expression and its plasma membrane association. In all three models (resting, lactating and suckling) there was an inverse correlation between extracellular copper concentration and the level of CTR1. Cell surface biotinylation studies demonstrated that as extracellular copper concentration increased membrane associated CTR1 was reduced. There was a significant increase in CTR1 expression (total and membrane associated) in the suckled gland model in comparison to the resting gland model, across all copper concentrations investigated (0-50 μM). Regulation of CTR1 expression was entirely post-translational, as quantitative real-time PCR analyses showed no change to CTR1 mRNA between all models and culture conditions. X-ray fluorescence microscopy on the differentiated PMC42-LA models revealed that organoid structures distinctively accumulated copper. Furthermore, as PMC42-LA cell cultures became progressively more specialised, successively more copper accumulated in organoids (resting<lactating<suckling), indicating a link between function and copper requirement. Based on previous data showing a function for CTR1 in copper uptake, we have concluded that under the influence of hormones and increased extracellular copper levels, CTR1 participates in uptake of copper by mammary epithelial cells, as a prerequisite for secretion of copper into milk.

Article	Year
Diesel exhaust particulate matter induces multinucleate cells and zinc transporter-dependent apoptosis in human airway cells. Immunology and cell biology\| PubMed ID: 17680010	2007
ATP7B expression in human breast epithelial cells is mediated by lactational hormones. The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society\| PubMed ID: 18180385	2008
Copper transport during lactation in transgenic mice expressing the human ATP7A protein. Biochemical and biophysical research communications\| PubMed ID: 18515074	2008
Bioinformatic and expression analyses of genes mediating zinc homeostasis in Nostoc punctiforme. Applied and environmental microbiology\| PubMed ID: 19011078	2009
Risk assessment in man and mouse. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 19188592	2009
The omega-3 fatty acid, DHA, decreases neuronal cell death in association with altered zinc transport. FEBS letters\| PubMed ID: 20006607	2010
A simultaneous temporal processing account of response rate. Behavioural processes\| PubMed ID: 20067825	2010
The importance of the reinforcer as a time marker. Behavioural processes\| PubMed ID: 20097275	2010
Reproductive toxic effects of sublethal cadmium on the marine polychaete Perinereis nuntia. Ecotoxicology and environmental safety\| PubMed ID: 20576290	2010
Antibiotics pollution in Jiulong River estuary: source, distribution and bacterial resistance. Chemosphere\| PubMed ID: 21620433	2011
Optimal temporal risk assessment. Frontiers in integrative neuroscience\| PubMed ID: 21991250	2011
Copper levels in buccal cells of vineyard workers engaged in various activities. The Annals of occupational hygiene\| PubMed ID: 22234616	2012
Copper and lactational hormones influence the CTR1 copper transporter in PMC42-LA mammary epithelial cell culture models. The Journal of nutritional biochemistry\| PubMed ID: 24485600	2013

David Freestone

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