Standardized and reproducible measurement of decision-making in mice.

Progress in science requires standardized assays whose results can be readily shared, compared, and reproduced across laboratories. Reproducibility, however, has been a concern in neuroscience, particularly for measurements of mouse behavior. Here, we show that a standardized task to probe decision-making in mice produces reproducible results across multiple laboratories. We adopted a task for head-fixed mice that assays perceptual and value-based decision making, and we standardized training protocol and experimental hardware, software, and procedures. We trained 140 mice across seven laboratories in three countries, and we collected 5 million mouse choices into a publicly available database. Learning speed was variable across mice and laboratories, but once training was complete there were no significant differences in behavior across laboratories. Mice in different laboratories adopted similar reliance on visual stimuli, on past successes and failures, and on estimates of stimulus prior probability to guide their choices. These results reveal that a complex mouse behavior can be reproduced across multiple laboratories. They establish a standard for reproducible rodent behavior, and provide an unprecedented dataset and open-access tools to study decision-making in mice. More generally, they indicate a path toward achieving reproducibility in neuroscience through collaborative open-science approaches.

In science, it is of vital importance that multiple studies corroborate the same result. Researchers therefore need to know all the details of previous experiments in order to implement the procedures as exactly as possible. However, this is becoming a major problem in neuroscience, as animal studies of behavior have proven to be hard to reproduce, and most experiments are never replicated by other laboratories. Mice are increasingly being used to study the neural mechanisms of decision making, taking advantage of the genetic, imaging and physiological tools that are available for mouse brains. Yet, the lack of standardized behavioral assays is leading to inconsistent results between laboratories. This makes it challenging to carry out large-scale collaborations which have led to massive breakthroughs in other fields such as physics and genetics. To help make these studies more reproducible, the International Brain Laboratory (a collaborative research group) et al. developed a standardized approach for investigating decision making in mice that incorporates every step of the process; from the training protocol to the software used to analyze the data. In the experiment, mice were shown images with different contrast and had to indicate, using a steering wheel, whether it appeared on their right or left. The mice then received a drop of sugar water for every correction decision. When the image contrast was high, mice could rely on their vision. However, when the image contrast was very low or zero, they needed to consider the information of previous trials and choose the side that had recently appeared more frequently. This method was used to train 140 mice in seven laboratories from three different countries. The results showed that learning speed was different across mice and laboratories, but once training was complete the mice behaved consistently, relying on visual stimuli or experiences to guide their choices in a similar way. These results show that complex behaviors in mice can be reproduced across multiple laboratories, providing an unprecedented dataset and open-access tools for studying decision making. This work could serve as a foundation for other groups, paving the way to a more collaborative approach in the field of neuroscience that could help to tackle complex research challenges.

Detectability of fifteen aquatic micro/mesocosms.

Zooplankton abundance and species richness in 15 untreated 12,000 L outdoor microcosms (n = 15) were monitored over the course of 1 year to document the inherent variability and statistical detectability between replicates. Statistical power analysis were applied to derive the statistically minimal detectable difference (MDD) between replicates with default values set at; alpha = 0.1 and beta = 0.2. Copepod abundance and species richness generally demonstrated the best detectability at 0.31 and 0.16, respectively, (n = 15); 0.59 and 0.33 (n = 3). Total zooplankton abundance and species richness had the lowest detectabilities at 0.19 and 0.14, respectively, (n = 15); 0.35 and 0.3 (n = 3). Rotifers, due to their opportunistic and rapid life traits, had the lowest single-species abundance detectabilities at 0.54 (n = 15); 0.8 (n = 3), whereas macroinvertebrate species richness had the lowest detectability at 0.43 (n = 15); 0.7 (n = 3) over 1 year. We recommend a priori calibration of the study design relative to relevant MDDs. Moreover, it is suggested to consider alternatives to statistical null hypothesis testing.

Toxicity and hazard of a mixture of SSRIs to zooplankton communities evaluated in aquatic microcosms.

The toxicity and hazard of a mixture of selective serotonin reuptake inhibitors (SSRIs), including fluoxetine, fluvoxamine, and sertraline, to zooplankton communities were evaluated using 120,00l outdoor microcosms. Acute (day 4) and chronic (day 35) zooplankton abundance and species richness were assessed for Rotifera, Cladocera, and Copepoda. For acute SSRI exposures, rotifers were the most sensitive zooplankton taxa to changes in abundance (predicted no effect concentration (PNEC)=19 nM); however, no effects in zooplankton species richness were observed for this treatment period. A decrease in Copepoda abundance and species richness was observed following chronic exposures of SSRIs (PNEC=9.1 nM). A 99th-centile predicted environmental concentration (PEC=0.51 nM) yielded HQs at least two orders of magnitude below 1. Therefore, mixtures of SSRIs do not appear to present a hazard to zooplankton communities at environmentally relevant concentrations.

Toxicity and hazard of selective serotonin reuptake inhibitor antidepressants fluoxetine, fluvoxamine, and sertraline to algae.

The toxicity of SSRIs to algae/phytoplankton was investigated using the US EPA ECOSAR, acute single-species growth inhibition assays, species sensitivity distributions (SSDs), and an outdoor microcosm mixture experiment. Worst-case ECOSAR estimates of SSRI toxicity to algae ranged from 0.73 to 13.08 mg/L. Sertraline was the most toxic SSRI tested in single-species growth inhibition assays followed by fluoxetine and fluvoxamine with worst-case 96-h IC10s of 4.6, 31.3, and 1662 microg/L, respectively. HC5s of 2.4, 3.6, and 1100 microg/L were estimated, respectively, for sertraline, fluoxetine, and fluvoxamine toxicity to algae-using SSDs. Microcosm phytoplankton structural endpoints were more sensitive than functional endpoints in the short term. However, in the long term, structural endpoints were resilient and functional endpoints remained impacted even after a period of recovery. The worst-case EC10 determined from the outdoor microcosm mixture toxicity to phytoplankton communities was 15.2 nM. Although SSRIs are toxic to algae, hazard quotients using worst-case PECs represent a margin of safety of 20 to phytoplankton. Although SSRIs do not appear to pose a hazard to primary production, this assessment is not protective of higher aquatic organisms and further research into the chronic toxicity to low levels of SSRIs to higher-level aquatic species is recommended.

Effects of a mixture of tetracyclines to Lemna gibba and Myriophyllum sibiricum evaluated in aquatic microcosms.

The impact of a mixture of oxytetracycline, chlortetracycline, tetracycline and doxycycline on Myriophyllum sibiricum and Lemna gibba was investigated using fifteen 12,000-L microcosms (k=5, n=3). Significant concentration-response relationships were only found for M. sibiricum, where dry mass was 69, 47, 30, and 7% of controls at respective treatment concentrations of 0.080, 0.218, 0.668, and 2.289 micromol/L. Somatic endpoints were strongly and negatively correlated with percent light transmission, except plant length, which was positively correlated. Treated microcosms experienced a reduction in the percent of surface irradiance penetrating the water column as high as 99.8% at a depth of 70 cm, relative to controls. Position relative to the water column was likely responsible for the differential effects observed between floating (L. gibba) and submerged (M. sibiricum) species of macrophytes. A hazard quotient assessment of the lowest EC10 value indicated significant risk, exceeding the critical HQ value, but not the lowest EC25 value.

Exposure assessment and microcosm fate of selected selective serotonin reuptake inhibitors.

The exposure and fate of selective serotonin reuptake inhibitors (SSRIs) was evaluated using modeled predicted environmental concentrations (PECs) according to the U.S. and the European Union (EU) guidelines and microcosm model ecosystems. According to the U.S. guidance, crude environmental introduction concentrations, the only SSRI that would require environmental assessment would be sertraline. However, the more conservative EU draft guidance PEC would require further assessment of all five SSRIs. Refined PECs developed using the U.S. and the EU guidelines along with estimates of removal by sewage treatment and receiving water dilution factors indicate that the U.S. methodology corresponds better to MEC data determined in the U.S. and Canada. Worst-case (99th centile) PECs for citalopram, fluoxetine, fluvoxamine, paroxetine, and sertraline were 30, 19, 30, 65, and 122 ng/L, respectively, using the U.S. methodology and 142, 182, 841, 144, and 575 ng/L, respectively, using the EU draft methodology. The dissipation of fluoxetine and fluvoxamine from the water column in aquatic microcosms was best described using a two-compartment model while sertraline followed a one-compartment model. Fluoxetine and fluvoxamine water concentrations initially dissipated with first phase half-lives of 3.8 and 1.8 days, respectively, but levelled off at concentrations around 10 microg/L with second phase half-lives of 76.7 and 59.3 days, respectively, not including those estimated as infinity. Sertraline dissipation tended toward the detection limit with a half-life of 3.4 days. Fluoxetine was found to be the most persistent followed by fluvoxamine and sertraline. Estimated log(K(OC)) values for all SSRIs were >4.3 indicating that SSRIs are expected to adsorb to sediment or sludge. Partitioning into other environmental compartments such as this may act as a reservoir from which SSRIs may be re-released into surface waters and indicates the potential susceptibility of benthos.

Dissipation of oxytetracycline, chlortetracycline, tetracycline and doxycycline using HPLC-UV and LC/MS/MS under aquatic semi-field microcosm conditions.

A mixture of four tetracyclines; oxytetracycline (OTC), chlortetracycline (CTC), tetracycline (TC), and doxycycline (DC) was applied in fifteen 12000l outdoor microcosms at four treatment levels plus controls each with three replicates (n = 3). The dissipation times of parent compounds were monitored and half-lives (DT50) of 1-4 days, depending on treatment level were recorded. This is in accordance with half-lives from previous findings in bench-top experiments. Parent compound DT50, were determined using HPLC-UV. Furthermore, the samples were analyzed for ten different tetracycline products using LC/MS/MS. Two products were found for chlortetracycline; 4-epi-anh-chlortetracyline and the iso-chlortetracycline. Iso-forms were only found for CTC and only at the highest treatment (300 microg l(-1)). The half-lives, trajectories, and relative amounts of the products were analogous for all four tetracyclines. DT50 for products were less than 1.2 days. Formation of 4-epi-anh-tetracyclines, occurred at neutral to weak alkaline conditions.

Aquatic microcosm assessment of the effects of tylosin on Lemna gibba and Myriophyllum spicatum.

Tylosin is a macrolide antibiotic commonly used for therapeutic treatment and prophylaxis in livestock. As part of a larger ecotoxicological study, the potential phytotoxic effects of tylosin on the rooted macrophyte Myriophyllum spicatum and the floating macrophyte Lemna gibba were assessed under semi-field conditions using 15 12 000-L microcosms. Concentrations of 0, 10, 30, 300 microg/L (n = 3), and 600, 1000, and 3000 microg/L (n = 1) were evaluated as part of separate ANOVA and regression analyses over an exposure period of 35 days. Fate of tylosin was monitored over time in the highest three treatments, where dissipation followed pseudo-first order kinetics with associated half-lives ranging from 9 to 10 days. For both M. spicatum and L. gibba, tylosin was found to cause no biologically significant changes to any endpoint assessed compared to controls at a Type I error rate of 0.1. However, subsequent power analyses revealed that there was generally insufficient power to declare that there were no significant differences at a Type II error rate of 0.2. Conclusions concerning biologically significant impacts were therefore further assessed based on other statistical criteria including comparisons of percent differences between replicated treatments and controls, minimum significant and minimum detectable differences, and coefficients of variation. Based on these criteria, at an ecological effect size of >20% change, tylosin was concluded to elicit no biologically or ecologically significant toxicity to M. spicatum or L. gibba. A hazard quotient assessment indicated that tylosin poses little risk to either species of macrophyte, with an HQ value calculated to be nearly three orders of magnitude below 1 (0.002).

Structural and functional responses of plankton to a mixture of four tetracyclines in aquatic microcosms.

Pharmaceuticals are routinely detected at low concentrations in surface waters, but effects on non-target organisms are not well understood. Microcosms were used to assess ecological responses in freshwater ecosystems to a mixture offourtetracyclines commonly used in veterinary and human medicine. Triplicate microcosms were treated with tetracycline, oxytetracycline, doxycycline, and chlortetracycline, resulting in measured time-weighted average total mixture concentrations of 0, 0.080, 0.218, 0.662, and 2.29 microM, respectively. Responses were assessed in terms of structure and function based on measurements of zooplankton and phytoplankton communities, ecosystem productivity, and water quality. Effects were observed for some endpoints > or = the 0.218 microM treatment. The largest responses were concentration-dependent reductions in total phytoplankton abundance and species richness. Phytoplankton abundance recovered to control levels in all microcosms after treatment was terminated, and resilience (time to return to normal operating range during stress) was observed with respectto phytoplankton species richness. Zooplankton were generally unaffected by the tetracyclines. Responses also included decreased water clarity, lower oxygen concentration, and water temperature. Functional endpoints showed varying sensitivity. On the basis of dissolved oxygen concentrations, community respiration (R) increased while primary productivity (P) was unchanged with increased treatment concentration. The effects observed occurred at considerably greater concentrations than are currently measured in the environment, indicating minimal risk to aquatic organisms.

Aquatic persistence of eight pharmaceuticals in a microcosm study.

The persistence of eight pharmaceuticals from multiple classes was studied in aquatic outdoor field microcosms. A method was developed for the determination of a mixture of acetaminophen, atorvastatin, caffeine, carbamazepine, levofloxacin, sertraline, sulfamethoxazole, and trimethoprim at microg/L levels from surface water of the microcosms using solid phase extraction and high-performance liquid chromatography-ultraviolet (HPLC-UV) and liquid chromatography tandem mass spectrometry (LC-MS-MS). Half-lives in the field ranged from 1.5 to 82 d. Laboratory persistence tests were performed to determine the relative importance of possible loss processes in the microcosms over the course of the study. Results from dark control experiments suggest hydrolysis was not important in the loss of the compounds. No significant differences were observed between measured half-lives of the pharmaceuticals in sunlight-exposed pond water and autoclaved pond water, which suggests photodegradation was important in limiting their persistence, and biodegradation was not an important loss process in surface water over the duration of the study. Observed photoproducts of several of the pharmaceuticals remained photoreactive, which led to further degradation in irradiated surface waters.

Toxicity classification and evaluation of four pharmaceuticals classes: antibiotics, antineoplastics, cardiovascular, and sex hormones.

Four different classes of environmental concern are quantitatively and qualitatively assessed for environmental hazards; antibiotics (n = 226), antineoplastics (n = 81), cardiovascular (n = 272), and sex hormones (n = 92). These along with an ECOSAR scan of all pharmaceuticals (n = 2848) were then classified according to the OECD aquatic toxicity classification system. The predicted species susceptibility is: daphnid > fish > algae, and the predicted rank order of relative toxicity: sex hormones > cardiovascular = antibiotics > antineoplastics (Table 1). Generally, a relatively large proportion (1/3) of all pharmaceuticals are potentially very toxic to aquatic organisms (Table 2). The qualitative risk assessment ranking relative to probability and potential severity for human and environmental health effects is: antibiotics > sex hormones > cardiovascular > antineoplastics. (Q)SARs and pharmacodynamic information should be used to prioritize and steer experimental risk assessments of pharmaceuticals, and potentially, also be used in new drug discovery optimizing efficacy and in minimising environmental hazards of new products. Nuclear receptors are relatively well conserved in evolution. Currently, antibacterial resistance represents the most significant human health hazard, and potentially the largest non-target organism hazard is sex hormones acting as endocrine modulators in wildlife. Data for the individual compounds are accessible via.

Effects of pharmaceutical mixtures in aquatic microcosms.

Pharmaceuticals have a wide range of biological properties and are released into the environment in relatively large amounts, yet little information is available regarding their effects or potential ecological risks. We exposed outdoor aquatic microcosms to combinations of ibuprofen (a nonsteroidal anti-inflammatory drug), fluoxetine (a selective serotonin reuptake inhibitor), and ciprofloxacin (a DNA gyrase-inhibiting antibiotic) at concentrations of 6, 10, and 10 microg/L, respectively (low treatment [LT]); 60, 100, and 100 microg/L, respectively (medium treatment [MT]); and 600, 1,000, and 1,000 microg/L, respectively (high treatment [HT]). We maintained these concentrations for 35 d. Few responses were observed in the LT; however, effects were observed in the MT and HT. Fish mortality occurred in the MT (<35 d) and in the HT (<4 d). Phytoplankton increased in abundance and decreased in diversity (number of taxa) in the HT, with consistent trends being observed in the MT and LT. Zooplankton also showed increased abundance and decreases in diversity in the HT, with consistent trends being observed in the MT. Multivariate analyses for zooplankton and phytoplankton suggested interactions between these communities. Lemna gibba and Myriophyllum spp. showed mortality in the HT; growth of L. gibba was also reduced in the MT. Bacterial abundance did not change in the HT. All responses were observed at concentrations well below the equivalent pharmacologically active concentrations in mammals. Although the present data do not suggest that ibuprofen, fluoxetine, and ciprofloxacin are individually causing adverse effects in surface-water environments, questions remain about additive responses from mixtures.

Ranking and prioritization of environmental risks of pharmaceuticals in surface waters.

Pharmaceuticals have been reported in surface waters, prompting legitimate public concern, as pharmaceuticals are biologically active compounds used daily by the public. Currently there are ecotoxicological data available for <1%, thus, the European Union Commission's Scientific committee on Toxicity, Ecotoxicity, and Environment (CSTEE) recommended use of (Q)SAR models and precaution to prioritize further risk assessment of approximately 4500 compounds and their adjuvants. We ranked 2986 different pharmaceutical compounds in 51 classes relative to hazard toward algae, daphnids, and fish using the EPIWIN program. This ranking cannot be used to acquit a compound based on predicted relative ranking. Modifying additives were the most toxic classes. Cardiovascular, gastrointestinal, antiviral, anxiolytic sedatives hypnotics and antipsychotics, corticosteroid, and thyroid pharmaceuticals were the predicted most hazardous therapeutic classes. The overall relative order of susceptibility was estimated to be daphnids > fish > algae. Expert judgment is needed to assess specific hazards for classes like microbial resistance and antibiotics, sex hormones, and endocrine disruptors. As human health and the function of ecological systems are interconnected and subject to the precautionary principle, harmonization of evidence for correlation and causality of adverse effects seems sensible in an ethical and cost-effective context to facilitate substitution of hazardous compounds. Data available: http://www.uoguelph.ca/~hsander/.

Response of the zooplankton community and environmental fate of perfluorooctane sulfonic acid in aquatic microcosms.

Little is known regarding perfluorooctane sulfonic acid (PFOS) toxicity to freshwater organisms. This field evaluation aims to assess the toxicological risk associated with exposure to PFOS across levels of biological organization. The analysis of variance study was conducted in replicate (n = 3) 12,000 L outdoor microcosms. Multivariate techniques were used to assess the response of zooplankton community structure and dynamics, as well as a floating macrophyte, Lemna gibba. The zooplankton community was significantly affected (p < 0.05) by the treatment regime given by the Monte Carlo permutations for all sampling times. A community-level no-observable-effect concentration ([NOEC]community) of 3.0 mg/L was determined for the 35-day study, however, longer term studies are recommended. The most sensitive taxonomic groups, Cladocera and Copepoda, were virtually eliminated in 30 mg/L treatments after 7 d. The 42-d 50% inhibition concentration (IC50) for L. gibba frond number was 19.1 mg/L and the NOEC was 0.2 mg/L. Furthermore, we investigated the persistence of PFOS over 285 d in microcosms under natural conditions. Perfluorooctane sulfonic acid concentration showed no drastic reduction in any treatment microcosm over the entire study period, confirming that this compound undergoes little degradation in aquatic systems. Presently, there appears to be little hazard to these freshwater organisms at reported environmental concentrations.

Probabilistic hazard assessment of environmentally occurring pharmaceuticals toxicity to fish, daphnids and algae by ECOSAR screening.

The risks associated with occurrence of pharmaceuticals in water resources are mostly unknown. In the absence of extensive toxicological data, we scanned all the compounds observed in the environment for toxicological properties by (Quantitative) Structure Activity Relationship ((Q)SAR). The results of the probabilistic distribution of environmental and effect concentrations and hazard quotients (HQs) do not indicate significant acute risks prior to application of assessment factors. Compared with measured effect concentrations SAR predictions were more "sensitive" 80% of the time. The long-term effects of subtle and chronic changes, additive or synergistic effects and effects on other endpoints e.g. reproduction, behavior, metabolism, bacterial resistance etc. are still uncertain. (Q)SAR's can be important prioritization tools for subsequent experimental risk assessment of pharmaceuticals in surface waters, due to the prevalent lack of ecotoxicological data.