Maternal antibiotic administration during a critical developmental window has enduring neurobehavioural effects in offspring mice.

Rates of perinatal maternal antibiotic use have increased in recent years linked to prophylactic antibiotic use following Caesarean section delivery. This antibiotic use is necessary and beneficial in the short-term; however, long-term consequences on brain and behaviour have not been studied in detail. Here, we endeavoured to determine whether maternal administration of antibiotics during a critical window of development in early life has lasting effects on brain and behaviour in offspring mice. To this end we studied two different antibiotic preparations (single administration of Phenoxymethylpenicillin at 31 mg/kg/day; and a cocktail consisting of, ampicillin 1 mg/mL; vancomycin 0.5 mg/mL; metronidazole 1 mg/mL; ciprofloxacin 0.2 mg/mL and imipenem 0.25 mg/mL). It was observed that early life exposure to maternal antibiotics led to persistent alterations in anxiety, sociability and cognitive behaviours. These effects in general were greater in animals treated with the broad-spectrum antibiotic cocktail compared to a single antibiotic with the exception of deficits in social recognition which were more robustly observed in Penicillin V exposed animals. Given the prevalence of maternal antibiotic use, our findings have potentially significant translational relevance, particularly considering the implications on infant health during this critical period and into later life.

In utero exposure to protease inhibitor-based antiretroviral regimens delays growth and developmental milestones in mice.

Antiretroviral therapy (ART) in pregnancy has dramatically reduced HIV vertical transmission rates. Consequently, there is a growing number of children that are HIV exposed uninfected (CHEUs). Studies suggest that CHEUs exposed in utero to ART may experience developmental delays compared to their peers. We investigated the effects of in utero ART exposure on perinatal neurodevelopment in mice, through assessment of developmental milestones. Developmental milestone tests (parallel to reflex testing in human infants) are reflective of brain maturity and useful in predicting later behavioral outcomes. We hypothesized that ART in pregnancy alters the in utero environment and thereby alters developmental milestone outcomes in pups. Throughout pregnancy, dams were treated with boosted-atazanavir combined with either abacavir/lamivudine (ATV/r/ABC/3TC), or tenofovir/emtricitabine (ATV/r/TDF/FTC), or water as control. Pups were assessed daily for general somatic growth and on a battery of tests for primitive reflexes including surface-righting, negative-geotaxis, cliff-aversion, rooting, ear-twitch, auditory-reflex, forelimb-grasp, air-righting, behaviors in the neonatal open field, and olfactory test. In utero exposure to either ART regimen delayed somatic growth in offspring and evoked significant delays in the development of negative geotaxis, cliff-aversion, and ear-twitch reflexes. Exposure to ATV/r/ABC/3TC was also associated with olfactory deficits in male and forelimb grasp deficits in female pups. To explore whether delays persisted into adulthood we assessed performance in the open field test. We observed no significant differences between treatment arm for males. In females, ATV/r/TDF/FTC exposure was associated with lower total distance travelled and less ambulatory time in the centre, while ATV/r/ABC/3TC exposure was associated with higher resting times compared to controls. In utero PI-based ART exposure delays the appearance of primitive reflexes that involve vestibular and sensory-motor pathways in a mouse model. Our findings suggest that ART could be disrupting the normal progress/maturation of the underlying neurocircuits and encourage further investigation for underlying mechanisms.

An Environmental Model of Honey Bee Colony Collapse Due to Pesticide Contamination.

We develop a model of honey bee colony collapse based on the contamination of forager bees in environmental regions contaminated with pesticides. An important feature of the model is the daily homing capacity each day of foragers bees. The model consists of difference equations describing the daily homing of uncontaminated and contaminated forager bees, with an increased homing failure of contaminated bees. The model quantifies colony collapse in terms of the fraction of contaminated bees subject to this increased homing failure. If the fraction is sufficiently high, then the hive falls below a viability threshold population size that leads to rapid disintegration. If the fraction is sufficiently low, then the hive can rise above the viability threshold and attain a stable population level.

Impact of acute oral exposure to thiamethoxam on the homing, flight, learning acquisition and short-term retention of Apis cerana.

BACKGROUND: Thiamethoxam (TMX) represents the second generation of neonicotinoids that has been widely applied in agricultural activities, while how TMX alters the behavior of Apis cerana, an important native honey bee species in China, is not clear. We carried out three independent experiments to study the impact of acute oral treatment of 20 µL TMX at concentrations of 2.4 ppb (0.048 ng/bee) and 10 ppb (0.2 ng/bee) on the homing, flight, learning acquisition and short-term retention ability of A. cerana. The homing ability was assessed by the catch-and-release method, the flight ability was assessed by flight mills, and the learning acquisition and short-term retention were evaluated by the proboscis extension response method. RESULTS: When treated with 10 ppb of TMX, bees had a significantly higher average homing time, mean flight velocity, flying distance, and flying duration than the control, whereas 2.4 ppb concentration did not cause any significant effect on homing or flight ability. Bees treated with either 2.4 ppb or 10 ppb TMX had significantly lower learning acquisition and short-term retention ability. CONCLUSION: Results suggest that acute oral exposure to 10 ppb of TMX altered the short-distance homing time, flight ability, and learning acquisition and short-term retention ability. Our study also highlights the concern that acute oral exposure to a low concentration of 2.4 ppb could have consequences on the behavior of A. creana. Those multiple sublethal alterations on A. cerana's behavior indicate that TMX are likely having complex but negative consequences on bee health in the field. © 2019 Society of Chemical Industry.

Hazard of a neonicotinoid insecticide on the homing flight of the honeybee depends on climatic conditions and Varroa infestation.

The paradigm for all toxicological bioassays in the risk assessment of pesticide registration reflects the principle that experimental conditions should be controlled to avoid any other factors that may affect the endpoint measures. As honeybee colonies can be frequently exposed to bio-aggressors in real conditions, often concomitantly with pesticides, co-exposure to pesticide/bio-aggressors is becoming a concern for regulatory authorities. We investigated the effects of the neonicotinoid insecticide thiamethoxam on the homing performances of foragers emerging from colonies differentiated by health status (infestation with Varroa destructor mites, microsporidian parasite Nosema spp. and Deformed Wing Virus). We designed a homing test that has been recently identified to fill a regulatory gap in the field evaluations of sublethal doses of pesticides before their registration. We also assessed the effect of temperature as an environmental factor. Our results showed that the Varroa mite exacerbates homing failure (HF) caused by the insecticide, whereas high temperatures reduce insecticide-induced HF. Through an analytical Effective Dose (ED) approach, predictive modeling results showed that, for instance, ED level of an uninfested colony, can be divided by 3.3 when the colony is infested by 5 Varroa mites per 100 bees and at a temperature of 24 °C. Our results suggest that the health status of honeybee colonies and climatic context should be targeted for a thorough risk assessment.

Short-Term Exposure to Lambda-Cyhalothrin Negatively Affects the Survival and Memory-Related Characteristics of Worker Bees Apis mellifera.

Pesticides are considered one of the major contemporary stressors of honey bee health. In this study, the effects of short-term exposure to lambda-cyhalothrin on lifespan, learning, and memory-related characteristics of Apis mellifera were systematically examined. Short-term exposure to lambda-cyhalothrin in worker bees reduced lifespan, affected learning and memory performance, reduced the homing ability, and influenced the expression levels of two learning and memory-related genes of A. mellifera. This research identifies the nature of the sublethal effects of lambda-cyhalothrin on bees and the level of exposure that can be harmful to bee health. This new information will assist in establishing guidelines for the safe use of lambda-cyhalothrin in the field.

The longitudinal effects of early developmental cadmium exposure on conditioned place preference and cardiovascular physiology in zebrafish.

Cadmium (Cd) is a naturally occurring trace metal that is widely considered to be highly toxic to aquatic organisms and a significant health hazard to humans (Amzal et al., 2009; Bernhoft 2013; Burger, 2008; Satarug et al., 2009). The zebrafish (Danio rerio) has been used as a model organism for toxicological studies with Cd (Banni et al., 2011; Blechinger et al., 2007; Chow et al., 2009; Chow et al., 2008; Favorito et al., 2011; Kusch et al., 2007; Matz et al., 2007; Wang and Gallagher, 2013). We asked what the lasting longitudinal effects would be from short early developmental Cd exposure (between 24 and 96h post-fertilization) in a range that larvae might experience living atop typical Cd-containing surface sediments (0, 0.01, 0.1, 1.0 and 10µM CdCl2: 1.124, 11.24, 112.4 and 1124µg Cd/L). The goal of this exposure window was to specifically target secondary neurogenesis, monoaminergic differentiation and cardiovascular development, without affecting earlier patterning processes. Developmental abnormalities in body size and CNS morphology increased with concentration, but were statistically significant only at the highest concentration used (10µM). Heart rate for Cd-treated larvae increased with concentration, and was significant even at the lowest concentration used (0.01µM). Longitudinal survival was significantly lower for fish developmentally exposed to the highest concentration. Except for brain weight, overall morphology was not affected by developmental Cd exposure. However, developmental exposure to lower concentrations of Cd (0.01, 0.1, and 1.0µM) progressively lowered cocaine-induced conditioned place preference (CPP), used to measure function of the reward pathways in the brain. Baseline heart rate was significantly lower in longitudinal fish developmentally exposed to 1.0µM Cd. Cardiovascular response to isoproterenol, a potent ß-adrenergic agonist, in longitudinal adults was also significantly affected by developmental exposure to Cd at low doses (0.01, 0.1 and 1.0µM). Surviving longitudinal adult fish exposed to the highest concentration of Cd showed normal CPP and cardiovascular physiology. The data imply that even lower exposure concentrations can potentially result in fitness-affecting parameters without affecting survival in a laboratory setting.

Homing pigeons externally exposed to Deepwater Horizon crude oil change flight performance and behavior.

The Deepwater Horizon oil spill was the largest in U.S. history, contaminating thousands of miles of coastal habitat and affecting the lives of many avian species. The Gulf of Mexico is a critical bird migration route area and migrants that were oiled but did not suffer mortality as a direct result of the spill faced unpredictable fates. This study utilized homing pigeons as a surrogate species for migratory birds to investigate the effects a single low level external oiling event has on the flight performance and behavior of birds flying repeated 161 km flights. Data from GPS data loggers showed that lightly oiled pigeons changed their flight paths, increased their flight durations by 2.6 fold, increased their flight distances by 28 km and subsequently decreased their route efficiencies. Oiled birds also exhibited reduced rate of weight gain between flights. Our data suggest that contaminated birds surviving the oil spill may have experienced flight impairment and reduced refueling abilities, likely reducing overall migration speed. Our findings contribute new information on how oil spills affect avian species, as the effects of oil on the flight behavior of long distance free-flying birds have not been previously described.

Body mass change in flying homing pigeons externally exposed to Deepwater Horizon crude oil.

The Deepwater Horizon oil spill contaminated thousands of miles of habitat valuable to hundreds of species of migratory and resident birds of the Gulf of Mexico. Many birds died as a direct result of the oil spill; however, the indirect effects of oil exposure on the flight ability and body condition of birds are difficult to assess in situ. This study utilizes the homing pigeon as a surrogate species for migratory birds to investigate the effect of multiple external oil exposures on the flight performance and body mass change of birds over a series of repeated flights from 136.8km flight distance. Oiled pigeons took significantly longer to return home, lost more weight during flight, and were unable to recover their weight, resulting in reduction of body weight overtime. Based on our data, migratory birds that were oiled, even partially, by the Deepwater Horizon oil spill likely took longer to complete migration and were likely in poor body condition, increasing their risk of mortality and reproductive failure.

The behavioural homing response of adult chum salmon Oncorhynchus keta to amino-acid profiles.

Adult chum salmon Oncorhynchus keta homing behaviour in a two-choice test tank (Y-maze) was monitored using a passive integrated transponder (PIT)-tag system in response to river-specific dissolved free amino-acid (DFAA) profiles and revealed that the majority of O. keta showed a preference for artificial natal-stream water and tended to stay in this maze arm for a longer period; natal-stream water was chosen over a nearby tributary's water, but not when the O. keta were presented with a non-tributary water. The results demonstrate the ability of O. keta to discriminate artificial stream waters containing natural levels of DFAA.

(R)-Salsolinol, a product of ethanol metabolism, stereospecifically induces behavioral sensitization and leads to excessive alcohol intake.

Ethanol is oxidized in the brain to acetaldehyde, which can condense with dopamine to generate (R/S)-salsolinol [(RS)-SAL]. Racemic salsolinol [(RS)-SAL] is self-infused by rats into the posterior ventral tegmental area (VTA) at significantly lower concentrations than those of acetaldehyde, suggesting that (RS)-SAL is a most active product of ethanol metabolism. Early studies showed that repeated intraperitoneal or intra-VTA administration of (RS)-SAL (10 mg/kg) induced conditioned place preference, led to locomotor sensitization and increased voluntary ethanol consumption. In the present study, we separated the (R)- and (S)-enantiomers from a commercial (RS)-SAL using a high-performance liquid chromatography with electrochemical detection system fitted with a ß-cyclodextrin-modified column. We injected (R)-SAL or (S)-SAL (30 pmol/1.0 µl) into the VTA of naïve UChB rats bred as alcohol drinkers to study whether one or both SAL enantiomers are responsible for the motivated behavioral effects, sensitization and increase in voluntary ethanol intake. The present results show that repeated administration of (R)-SAL leads to (1) conditioned place preference; (2) locomotor sensitization; and (3) marked increases in binge-like ethanol intake. Conversely, (S)-SAL did not influence any of these parameters. Overall, data indicate that (R)-SAL stereospecifically induces motivational effects, behavioral sensitization and increases ethanol intake.

Acquisition of morphine conditioned place preference increases the dendritic complexity of nucleus accumbens core neurons.

Contexts associated with opioid reward trigger craving and relapse in opioid addiction. Effects of reward-context associative learning on nucleus accumbens (NAc) dendritic morphology were studied using morphine conditioned place preference (CPP). Morphine-conditioned mice received saline and morphine 10 mg/kg subcutaneous (s.c.) on alternate days. Saline-conditioned mice received saline s.c. each day. Morphine-conditioned and saline-conditioned groups received injections immediately before each of eight daily conditioning sessions. Morphine homecage controls had no CPP training, but received saline and morphine in the homecage concomitantly with the morphine-conditioned group. Morphine conditioning produced greater place preference than saline conditioning. Mice were sacrificed 1 day after CPP expression. Dendritic changes were studied using Golgi-Cox staining and digital tracing of NAc core and shell neurons. In the NAc core, morphine homecage administration increased spine density, while morphine conditioning increased dendritic complexity, as defined by increased dendritic count, length and intersections. Place preference positively correlated with dendritic length and intersections in the NAc core. The core may mediate reward consolidation and determine how context-related signals from the shell lead to motor behavior. The combination of drug and conditioning in the morphine-conditioned group produced unique morphological effects different from the effects of drug or conditioning procedures by themselves. An additional study found no differences in neuron morphology between saline-conditioned mice, trained as described earlier, and mice that were not conditioned, but received saline in the homecage. The unique effect of morphine reward learning on NAc core dendrites reflects a brain substrate that could be targeted for therapeutic intervention in addiction.

Effects of sublethal doses of glyphosate on honeybee navigation.

Glyphosate (GLY) is a herbicide that is widely used in agriculture for weed control. Although reports about the impact of GLY in snails, crustaceans and amphibians exist, few studies have investigated its sublethal effects in non-target organisms such as the honeybee Apis mellifera, the main pollen vector in commercial crops. Here, we tested whether exposure to three sublethal concentrations of GLY (2.5, 5 and 10 mg l(-1): corresponding to 0.125, 0.250 and 0.500 µg per animal) affects the homeward flight path of honeybees in an open field. We performed an experiment in which forager honeybees were trained to an artificial feeder, and then captured, fed with sugar solution containing traces of GLY and released from a novel site either once or twice. Their homeward trajectories were tracked using harmonic radar technology. We found that honeybees that had been fed with solution containing 10 mg l(-1) GLY spent more time performing homeward flights than control bees or bees treated with lower concentrations. They also performed more indirect homing flights. Moreover, the proportion of direct homeward flights performed after a second release from the same site increased in control bees but not in treated bees. These results suggest that, in honeybees, exposure to levels of GLY commonly found in agricultural settings impairs the cognitive capacities needed to retrieve and integrate spatial information for a successful return to the hive. Therefore, honeybee navigation is affected by ingesting traces of the most widely used herbicide worldwide, with potential long-term negative consequences for colony foraging success.

Effects of the juvenile hormone analogue methoprene on rate of behavioural development, foraging performance and navigation in honey bees (Apis mellifera).

Worker honey bees change roles as they age as part of a hormonally regulated process of behavioural development that ends with a specialised foraging phase. The rate of behavioural development is highly plastic and responsive to changes in colony condition such that forager losses, disease or nutritional stresses accelerate behavioural development and cause an early onset of foraging in workers. It is not clear to what degree the behavioural development of workers can be accelerated without there being a cost in terms of reduced foraging performance. Here, we compared the foraging performance of bees induced to accelerate their behavioural development by treatment with the juvenile hormone analogue methoprene with that of controls that developed at a normal rate. Methoprene treatment accelerated the onset of both flight and foraging behaviour in workers, but it also reduced foraging span, the total time spent foraging and the number of completed foraging trips. Methoprene treatment did not alter performance in a short-range navigation task, however. These data indicate a limitation to the physiological plasticity of bees, and a trade off between forager performance and the speed at which bees begin foraging. Chronic stressors will be expected to reduce the mean age of the foraging force, and therefore also reduce the efficiency of the foraging force. This interaction may explain why honey bee colonies react to sustained stressors with non-linear population decline.

Evaluation of social and nonsocial behaviors mediated by opioids in mouse pups.

The experimental approach to carry out a behavioral study involving opioids in mouse pups needs equipments and procedures different from those used for adult animals. Pups are immature at birth and only slowly acquire all the potentialities that characterize adult con-specifics. The standard and abnormal development of behavioral systems and their neural correlates can be followed during the first postnatal weeks, using appropriate methodologies that exploit characteristic pups' capabilities. Behavioral tests designed for pups to evaluate the activity and involvement of the opioid system, according to the well-known role of the system in adult animals, are described in this chapter.

Effects of perinatal methylphenidate (MPH) treatment on postweaning behaviors of male and female Sprague-Dawley rats.

Methylphenidate (MPH) is a common treatment for adult Attention Deficit Hyperactivity Disorder (ADHD). However, little information exists regarding its safety during pregnancy and thus, women with ADHD face difficult decisions regarding continued use during pregnancy. Thus, Sprague-Dawley rats were orally treated 3×/day with 0 (control), 6 (low), 18 (mid), or 42 (high) mg MPH/kg/day (i.e., 0, 2, 6, or 14mg/kg at each treatment time) on gestational days 6-21. All offspring/litter were orally treated with the same dose their dam had received on postnatal days (PNDs) 1-21. After weaning, offspring were assessed for adolescent play behavior, locomotor activity, motor coordination, Barnes maze performance, acoustic startle response, novel object recognition, residential running wheel activity, flavored solution intake, home cage behavior, water maze performance, elevated plus maze behavior, locomotor response to an MPH challenge, and passive avoidance. At euthanasia, whole brain and striatal weights as well as serum hormone levels were measured. Body weights of the high MPH group were reduced in both sexes. Males of the high MPH group were less active than control males in open field assessments on PNDs 40-42. Latency to maximum acoustic startle was significantly altered in females of the medium and high MPH groups and residential running wheel activity of females of the low and medium MPH groups was lower than control females. Open arm entries in the elevated plus maze were increased in subjects of the medium MPH group. Females of the low MPH group were less sensitive to the locomotor-increasing effects of an acute 5mg/kg MPH challenge. Serum hormone levels and whole brain and striatal weights were not altered by prior MPH treatment. These results indicate that MPH treatment during development has sporadic effects on postweaning behaviors and those effects were generally exhibited by females.

Pesticide risk assessment in free-ranging bees is weather and landscape dependent.

The risk assessment of plant protection products on pollinators is currently based on the evaluation of lethal doses through repeatable lethal toxicity laboratory trials. Recent advances in honeybee toxicology have, however, raised interest on assessing sublethal effects in free-ranging individuals. Here, we show that the sublethal effects of a neonicotinoid pesticide are modified in magnitude by environmental interactions specific to the landscape and time of exposure events. Field sublethal assessment is therefore context dependent and should be addressed in a temporally and spatially explicit way, especially regarding weather and landscape physiognomy. We further develop an analytical Effective Dose (ED) framework to help disentangle context-induced from treatment-induced effects and thus to alleviate uncertainty in field studies. Although the ED framework involves trials at concentrations above the expected field exposure levels, it allows to explicitly delineating the climatic and landscape contexts that should be targeted for in-depth higher tier risk assessment.

Neonicotinoids interfere with specific components of navigation in honeybees.

Three neonicotinoids, imidacloprid, clothianidin and thiacloprid, agonists of the nicotinic acetylcholine receptor in the central brain of insects, were applied at non-lethal doses in order to test their effects on honeybee navigation. A catch-and-release experimental design was applied in which feeder trained bees were caught when arriving at the feeder, treated with one of the neonicotinoids, and released 1.5 hours later at a remote site. The flight paths of individual bees were tracked with harmonic radar. The initial flight phase controlled by the recently acquired navigation memory (vector memory) was less compromised than the second phase that leads the animal back to the hive (homing flight). The rate of successful return was significantly lower in treated bees, the probability of a correct turn at a salient landscape structure was reduced, and less directed flights during homing flights were performed. Since the homing phase in catch-and-release experiments documents the ability of a foraging honeybee to activate a remote memory acquired during its exploratory orientation flights, we conclude that non-lethal doses of the three neonicotinoids tested either block the retrieval of exploratory navigation memory or alter this form of navigation memory. These findings are discussed in the context of the application of neonicotinoids in plant protection.

Alterations in the rate of binge ethanol consumption: implications for preclinical studies in mice.

The rate at which alcohol (ethanol) is consumed has direct impact on its behavioral and subjective effects. For this reason, alterations in the pattern of ethanol consumption as a function of drinking history might be critical to the development and maintenance of alcoholism. Furthermore, because pharmacological interventions aimed at disrupting the motivation to consume ethanol are dependent on the brain/plasma concentrations present when an individual is most likely to engage in consumption of this substance, characterizing temporal drinking patterns might be useful to determine the timing of such treatments. The primary goal of the present study was to evaluate alterations in the timecourse of daily binge (drinking-in-the-dark; DID) ethanol consumption. We gave 14 daily 2 hour DID ethanol or water access sessions to male C57BL/6J (B6) mice using a state of the art volumetric drinking monitoring device. We then, primarily as a proof-of-principle, used the GABAB allosteric modulator GS39783 (GS) to determine how this compound influenced the timecourse of binge-like ethanol intake. The rate of ethanol consumption increased dramatically over sessions with the majority occurring in the first few minutes of the final session. Additionally, ethanol consumption occurring immediately following access was almost completely abolished in mice pre-treated with GS; an effect which was ethanol-specific only at this early time interval. These data characterize progressive alterations in the rate of ethanol intake using the DID model and suggest that careful consideration of prior ethanol history and timing of drug administration are warranted when interpreting results of pre-clinical drug administration studies.