Behavioral Endpoints Research Articles

A novel open-source raspberry Pi-based behavioral testing in zebrafish.

The zebrafish (Danio rerio) is widely used as a promising high-throughput model organism in neurobehavioral research. The mobility of zebrafish can be dissected into multiple behavior endpoints to assess its neurobehavioral performance. However, such facilities on the market are expensive and clumsy to be used in laboratories. Here, we designed a low-cost, automatic zebrafish behavior assay apparatus, barely without unintentional human operational errors. The data acquisition part, composed of Raspberry Pi and HQ Camera, automatically performs video recording and data storage. Then, the data processing process is also on the Raspberry Pi. Water droplets and inner wall reflection of multi-well cell culture plates (used for placing zebrafish) will affect the accuracy of object recognition. And during the rapid movement of zebrafish, the probability of zebrafish tracking loss increased significantly. Thus, ROI region and related thresholds were set, and the Kalman filter algorithm was performed to estimate the best position of zebrafish in each frame. In addition, all functions of this device are realized by the custom-written behavior analysis algorithm, which makes the optimization of the setup more efficient. Furthermore, this setup was also used to analyze the behavioral changes of zebrafish under different concentrations of alcohol exposure to verify the reliability and accuracy. The alcohol exposure induced an inverted U-shape dose-dependent behavior change in zebrafish, which was consistent with previous studies, showcasing that the data obtained from the setup proposed in this study are accurate and reliable. Finally, the setup was comprehensively assessed by evaluating the accuracy of zebrafish detection (precision, recall, F-score), and predicting alcohol concentration by XGBoost. In conclusion, this study provides a simple, and low-cost package for the determination of multiple behavioral parameters of zebrafish with high accuracy, which could be easily adapted for various other fields.

Adult and regenerating planarians respond differentially to chronic drug exposure

There is a lack of data on the effects of chronic exposure to common drugs and stimulants on the developing nervous system. Freshwater planarians have emerged as a useful invertebrate model amenable to high-throughput behavioral phenotyping to assay chemical safety in adult and developing brains. Here, we leverage the unique strength of the system to test in parallel for effects on the adult and developing nervous system, by screening ten common drugs and stimulants (forskolin, clenbuterol, LRE-1, MDL-12,330A, adenosine, caffeine, histamine, mianserin, fluoxetine and sertraline) using the asexual freshwater planarian Dugesia japonica. The compounds were tested up to 100 μM nominal concentration for their effects on planarian morphology and behavior. Quantitative phenotypic assessments were performed on days 7 and 12 of exposure using an automated screening platform. The antidepressants sertraline and fluoxetine were the most potent to induce lethality, with significant lethality observed at 10 μM. All ten compounds caused sublethal morphological and/or behavioral effects, with the most effects, in terms of potency and breadth of endpoints affected, seen with mianserin and fluoxetine. Four of the compounds (forskolin, clenbuterol, mianserin, and fluoxetine) were developmentally selective, causing effects at lower concentrations in regenerating planarians. Of these, fluoxetine showed the greatest differences between the two developmental stages, inducing many behavioral endpoints in regenerating planarians but only a few in adult planarians. While some of these behavioral effects may be due to neuroefficacy, these results substantiate the need for better evaluation of the safety of these common drugs on the developing nervous system.

Microglial P2Y12 mediates chronic stress-induced synapse loss in the prefrontal cortex and associated behavioral consequences.

Chronic unpredictable stress (CUS) drives microglia-mediated neuronal remodeling and synapse loss in the prefrontal cortex (PFC), contributing to deficits in cognition and behavior. However, it remains unclear what mechanisms guide microglia-neuron interactions in stress. Evidence indicates that neuronal activity-dependent purinergic signaling directs microglial processes and synaptic engagement via P2Y12, a purinergic receptor exclusively expressed by microglia in the brain. Stress alters excitatory neurotransmission in the PFC, thus we aimed to determine if P2Y12 signaling promotes functional changes in microglia in chronic stress. Here we used genetic ablation of P2Y12 (P2ry12-/-) or pharmacological blockade (clopidogrel, ticagrelor) to examine the role of purinergic signaling in stress-induced microglia-neuron interaction. Multiple behavioral, physiological, and cytometric endpoints were analyzed. Deletion of P2Y12 led to a number of fundamental alterations in the PFC, including the heightened microglial number and increased dendritic spine density. Flow cytometry revealed thatmicroglia inP2ry12-/- mice had shifts in surface levelsof CX3CR1, CSF1R, and CD11b, suggesting changes in synaptic engagement and phagocytosis in the PFC. In line with this, pharmacological blockade of P2Y12 prevented CUS-induced increases in the proportion of microglia with neuronal inclusions, limited dendritic spine loss in the PFC, and attenuated alterations in stress coping behavior and working memory function. Overall, these findings indicate that microglial P2Y12 is a critical mediator of stress-induced synapse loss in the PFC and subsequent behavioral deficits.

Early life exposure to high fructose diet induces metabolic dysregulation associated with sex-specific cognitive impairment in adolescent rats

The incidence of adolescent mental health disorders is on the rise. Epidemiological studies suggest that poor nutrition is a significant contributor to this public health crisis, specifically through exposure to high level of dietary sugar, including fructose, during critical periods of development. Previous studies have shown that elevated fructose exposure during adolescence disrupts mental health. Despite these data, it is currently unknown how fructose exposure, specifically during infancy, may impact adolescent mental health. We developed a rat experimental protocol to investigate the effects of fructose exposure during infancy on behavioral, cognitive and metabolic endpoints in adolescence. We found that exposing rats to high fructose from birth to weaning resulted in higher circulating glucose, insulin and leptin levels in adolescence. High fructose during infancy also increased bodyweight, disrupted metabolic homeostasis in the basolateral amygdala (BLA) as indicated by decreased activity of the cellular energy sensor AMPK, and impaired attention and impulsivity in a male-specific manner. This impaired attention observed in adolescent male rats following neonatal fructose exposure was partially rescued by viral-mediated, in vivo expression of a constitutively active form of AMPK in principal neurons of the BLA. Our results suggest that exposure to high level of fructose during infancy may impact adolescent mental health in a male-specific manner and that manipulation of AMPK activity may mitigate this impact.

Developmental Methylmercury Exposure Induced and Age-Dependent Glutamatergic Neurotoxicity in Caenorhabditis elegans.

Developmental methylmercury (MeHg) exposures cause latent neurotoxic effects in adults; however, the mechanisms underlying the latent neurotoxicity are not fully understood. In the current study, we used C. elegans as an animal model to investigate the latent neurotoxic effects of developmental MeHg exposures on glutamatergic neurons. The young larvae stage 1 worms were exposed to MeHg (0.05 ~ 5 µM) for 48h. The morphological and behavioral endpoints of glutamatergic neurons were compared when worms reached to adult stages including the young adult stage (day 1 adult) and the old adult stage (day 10 adult). Here, we showed that C. elegans glutamatergic neurons were morphologically intact following low or medium MeHg exposures (0.05 ~ 0.5 µM). The morphological damage of glutamatergic neurons appeared to be pronounced in day 10 adults developmentally exposed to 5 µM MeHg. Behavioral assays also showed an age-dependent latent effect of MeHg. In the nose touch response assay, only day 10 adult worms exhibited a functional decline following prior 5 µM MeHg exposure. Moreover, the disruption of NaCl memory appeared only in day 1 adults following MeHg exposures but not in day 10 adults. The expression of C. elegans homologs of mammalian vesicular glutamate transporter (eat-4) was repressed in day 1 adults, while the glutamate receptor homolog (glr-1) was upregulated in day 10 adults with 5 µM MeHg. In the comparison of age-dependent changes in the insulin-like pathway (daf-2/age-1/daf-16) following MeHg exposures, we showed that the daf-2/age-1/daf-16 pathway was mobilized in day 1 adults but repressed in day 10 adults. Collectively, our data supports a conclusion that MeHg-induced glutamatergic neurotoxicity exhibits an age-dependent pattern, possibly related to the prominent changes in age-dependent modulation in the glutamatergic neurotransmission and metabolic pathways.

Neurochemical, histological, and behavioral profiling of the acute, sub-acute, and chronic MPTP mouse model of Parkinson's disease.

Parkinson's disease (PD) is a heterogeneous multi-systemic disorder unique to humans characterized by motor and non-motor symptoms. Preclinical experimental models of PD present limitations and inconsistent neurochemical, histological, and behavioral readouts. The 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD is the most common in vivo screening platform for novel drug therapies; nonetheless, behavioral endpoints yielded amongst laboratories are often discordant and inconclusive. In this study, we characterized neurochemically, histologically, and behaviorally three different MPTP mouse models of PD to identify translational traits reminiscent of PD symptomatology. MPTP was intraperitoneally (i.p.) administered in three different regimens: (i) acute-four injections of 20 mg/kg of MPTP every 2 h; (ii) sub-acute-one daily injection of 30 mg/kg of MPTP for 5 consecutive days; and (iii) chronic-one daily injection of 4 mg/kg of MPTP for 28 consecutive days. A series of behavioral tests were conducted to assess motor and non-motor behavioral changes including anxiety, endurance, gait, motor deficits, cognitive impairment, circadian rhythm and food consumption. Impairments in balance and gait were confirmed in the chronic and acute models, respectively, with the latter showing significant correlation with lesion size. The sub-acute model, by contrast, presented with generalized hyperactivity. Both, motor and non-motor changes were identified in the acute and sub-acute regime where habituation to a novel environment was significantly reduced. Moreover, we report increased water and food intake across all three models. Overall, the acute model displayed the most severe lesion size, while across the three modelsstriatal dopamine content (DA) did not correlate with the behavioral performance. The present study demonstrates that detection of behavioral changes following MPTP exposure is challenging and does not correlate with the dopaminergic lesion extent.

Raloxifene Mitigates Emotional Deficits after Mild Traumatic Brain Injury in Mice.

Persons with mild traumatic brain injury (TBI) often exhibit persistent emotional impairments, particularly depression, fearfulness, and anxiety, that significantly diminish quality of life. Studying these mood disorders in animal models of mild TBI can help provide insight into possible therapies. We have previously reported that mice show increased depression, fearfulness, and anxiety, as well as visual and motor deficits, after focal cranial blast and that treatment with the cannabinoid type 2 receptor (CB2) inverse agonist, SMM-189, reduces these deficits. We have further shown that raloxifene, which is U.S. Food and Drug Administration approved as an estrogen receptor modulator to treat osteoporosis, but also possesses CB2 inverse agonism, yields a similar benefit for visual deficits in this model of TBI. Here, we have extended our studies of raloxifene benefit and show that it similarly reverses depression, fearfulness, and anxiety after focal cranial blast TBI in mice, using standard assays of these behavioral end-points. These results indicate the potential of raloxifene in the broad rescue of deficits after mild TBI and support phase 2 efficacy testing in human clinical trials.

SAHmap: Synergistic-antagonistic heatmap to evaluate the combined synergistic effect of mixtures of three pesticides on multiple endpoints of Caenorhabditis elegans

The environmental pollution caused by toxic chemicals such as pesticides has become a global problem. The mixture of dichlorvos (DIC), dimethoate (DIM), aldicarb (ALD) poses potential risks to the environment and human health. To fully explore the interaction of complex mixtures on Caenorhabditis elegans behavioral toxicity endpoint. This study created a synergistic-antagonistic heatmap (SAHmap) based on the combination index to systematically describe the toxicological interaction prospect of the mixture system. It was shown that the three pesticides and their binary as well as ternary mixture rays have significant concentration-response relationship on three behavioral endpoints of nematodes, From the perspective of synergistic-antagonistic heatmaps, all the mixture rays in the DIC-DIM mixture system showed strong synergism on the three behavioral and lethal endpoints. In the ternary mixture system, the five mixture rays showed different interaction between the behavioral endpoint and the lethal endpoint, and showed slight synergism to two behavioral endpoints as a whole. The emergence of synergism should arouse our attention to these hazardous chemicals. In addition, the use of SAHmap and the significant linear correlation among three behavioral endpoints further improved the efficiency of the study on the behavioral toxicity of pesticide mixtures to Caenorhabditis elegans.

Effects of cannabinoid exposure on short-term memory and medial orbitofrontal cortex function and chemistry in adolescent female rhesus macaques.

AimThere is increasing concern that cannabinoid exposure during adolescence may disturb brain maturation and produce long-term cognitive deficits. However, studies in human subjects have provided limited evidence for such causality. The present study utilized behavioral and neuroimaging endpoints in female non-human primates to examine the effects of acute and chronic exposure during adolescence to the cannabinoid receptor full agonist, AM2389, on cognitive processing and brain function and chemistry.Materials and methodsAdolescent female rhesus macaques were trained on a titrating-delay matching-to-sample (TDMTS) touchscreen task that assays working memory. TDMTS performance was assessed before and during chronic exposure to AM2389, following antagonist (rimonabant) administration, and after discontinuation of the chronic regimen. Resting-state fMRI connectivity and magnetic resonance spectroscopy data were acquired prior to drug treatment, during chronic exposure, and following its discontinuation. Voxels were placed in the medial orbitofrontal cortex (mOFC), a region involved in memory processing that undergoes maturation during adolescence.ResultsTDMTS performance was dose-dependently disrupted by acute AM2389; however, chronic treatment resulted in tolerance to these effects. TDMTS performance also was disrupted by discontinuation of the chronic regimen but surprisingly, not by rimonabant administration during chronic AM2389 treatment. mOFC N-acetylaspartate/creatine ratio decreased after acute and chronic administration but returned to baseline values following discontinuation of chronic treatment. Finally, intra-network functional connectivity (mOFC) increased during the chronic regimen and returned to baseline values following its discontinuation.ConclusionNeural effects of a cannabinergic drug may persist during chronic exposure, notwithstanding the development of tolerance to behavioral effects. However, such effects dissipate upon discontinuation, reflecting the restorative capacity of affected brain processes.

Bioactivation and detoxification of organophosphorus pesticides in freshwater planarians shares similarities with humans.

Organophosphorus pesticides (OPs) are a chemically diverse class of insecticides that inhibit acetylcholinesterase (AChE). Many OPs require bioactivation to their active oxon form via cytochrome P450 to effectively inhibit AChE. OP toxicity can be mitigated by detoxification reactions performed by carboxylesterase and paraoxonase. The relative extent of bioactivation to detoxification varies among individuals and between species, leading to differential susceptibility to OP toxicity. Because of these species differences, it is imperative to characterize OP metabolism in model systems used to assess OP toxicity. We have shown that the asexual freshwater planarian Dugesia japonica is a suitable model to assess OP neurotoxicity and developmental neurotoxicity via rapid, automated testing of adult and developing organisms in parallel using morphological and behavioral endpoints. D. japonica has two cholinesterase enzymes with intermediate properties between AChE and butyrylcholinesterase that are sensitive to OP inhibition. Here, we demonstrate that D. japonica contains the major OP metabolic machinery to be a relevant model for OP neurotoxicity studies. Adult and regenerating D. japonica can bioactivate chlorpyrifos and diazinon into their respective oxons. Significant AChE inhibition was only observed after in vivo metabolic activation but not when the parent OPs were directly added to planarian homogenate using the same concentrations and timing. Using biochemical assays, we found that D. japonica has both carboxylesterase (24nmol/(min*mg protein)) and paraoxonase (60pmol/(min*mg protein)) activity. We show that planarian carboxylesterase activity is distinct from cholinesterase activity using benzil and tacrine. These results further support the use of D. japonica for OP toxicity studies.

The Vasopressin 1a Receptor Antagonist SRX246 Reduces Aggressive Behavior in Huntington's Disease.

SRX246, an orally available CNS penetrant vasopressin (VP) V1a receptor antagonist, was studied in Huntington’s disease (HD) patients with irritability and aggressive behavior in the exploratory phase 2 trial, Safety, Tolerability, and Activity of SRX246 in Irritable HD patients (STAIR). This was a dose-escalation study; subjects received final doses of 120 mg BID, 160 mg BID, or placebo. The compound was safe and well tolerated. In this paper, we summarize the results of exploratory analyses of measures of problematic behaviors, including the Cohen–Mansfield Agitation Inventory (CMAI), Aberrant Behavior Checklist (ABC), Problem Behaviors Assessment-short form (PBA-s), Irritability Scale (IS), Clinical Global Impression (CGI), HD Quality of Life (QoL), and Caregiver Burden questionnaires. In addition to these, we asked subjects and caregivers to record answers to short questions about mood, irritability, and aggressive conduct in an eDiary. STAIR was the first rigorously designed study of behavioral endpoints like these in HD. The exploratory analyses showed that SRX246 reduced aggressive acts. Readily observed behaviors should be used as trial endpoints.

Concomitant medication use in children with autism spectrum disorder: Data from the Autism Biomarkers Consortium for Clinical Trials.

Children with autism spectrum disorder are prescribed a variety of medications that affect the central nervous system (psychotropic medications) to address behavior and mood. In clinical trials, individuals taking concomitant psychotropic medications often are excluded to maintain homogeneity of the sample and prevent contamination of biomarkers or clinical endpoints. However, this choice may significantly diminish the clinical representativeness of the sample. In a recent multisite study designed to identify biomarkers and behavioral endpoints for clinical trials (the Autism Biomarkers Consortium for Clinical Trials), school-age children with autism spectrum disorder were enrolled without excluding for medications, thus providing a unique opportunity to examine characteristics of psychotropic medication use in a research cohort and to guide future decisions on medication-related inclusion criteria. The aims of the current analysis were (1) to quantify the frequency and type of psychotropic medications reported in school-age children enrolled in the ABC-CT and (2) to examine behavioral features of children with autism spectrum disorder based on medication classes. Of the 280 children with autism spectrum disorder in the cohort, 42.5% were taking psychotropic medications, with polypharmacy in half of these children. The most commonly reported psychotropic medications included melatonin, stimulants, selective serotonin reuptake inhibitors, alpha agonists, and antipsychotics. Descriptive analysis showed that children taking antipsychotics displayed a trend toward greater overall impairment. Our findings suggest that exclusion of children taking concomitant psychotropic medications in trials could limit the clinical representativeness of the study population, perhaps even excluding children who may most benefit from new treatment options.

Digital Video Acquisition and Optimization Techniques for Effective Animal Tracking in Behavioral Ecotoxicology.

Behavioral phenotypic analysis is an emerging and increasingly important toolbox in aquatic ecotoxicology. In this regard digital video recording has recently become a standard in obtaining behavioral data. Subsequent analysis requires applications of specialized software for detecting and reconstructing animal locomotory trajectories as well as extracting quantitative biometric endpoints associated with specific behavioral traits. Despite some profound advantages for behavioral ecotoxicology, there is a notable lack of standardization of procedures and guidelines that would aid in consistently acquiring high-quality digital videos. The latter are fundamental for using animal tracking software successfully and to avoid issues such as identification switching, incorrect interpolation, and low tracking visibility. Achieving an optimized tracking not only saves user time and effort to analyze the results but also provides high-fidelity data with minimal artifacts. In the present study we, for the first time, provide an easily accessible guide on how to set up and optimize digital video acquisition while minimizing pitfalls in obtaining the highest-quality data for subsequent animal tracking. We also discuss straightforward digital video postprocessing techniques that can be employed to further enhance tracking consistency or improve the videos that were acquired in otherwise suboptimal settings. The present study provides an essential guidebook for any aquatic ecotoxicology studies that utilize digital video acquisition systems for evaluation of behavioral endpoints. Environ Toxicol Chem 2022;41:2342-2352. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Pharmacological approaches in the treatment of autism: a narrative review

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by deficits in communication and social interaction and by the presence of repetitive and restricted behaviors. To review the literature on drug interventions in the treatment of ASD in children and adolescents. Narrative review conducted at a university in São Paulo, Brazil. Search of the literature of studies published from 2005 to 2022, in Portuguese, English and Spanish, in the SciELO, PubMed, SCOPUS, LILACS and TripDatabase databases. Risperidone alone improved stereotyped patterns of behavior. Associations with aripiprazole or galantamine reduced irritability and managing the behavioral symptoms, respectively. Ginkgo biloba, associated to risperidone, led to an improvement in the Aberrant Behavior Checklist-Community (ABC-C) subscales scores. The association with sulforaphane exhibited a greater improvement in irritability and hyperactivity/noncompliance scores. Omega-3s reduced gesture use and hyperactivity. Bumetanide reduced restricted interests and repetitive behavior compared to placebo. Intranasal oxytocin improved social skills. The AB-2004 showed improvement in behavioral endpoints, especially anxiety and irritability. Folinic acid improved verbal communication in non-syndromic ASD children. Prednisolone increased language scores. There is not a drug that treats all of them and the possibility of significant adverse effects must be adjusted according to the symptoms and clinical response.

Behavioral and physiological toxicity thresholds of a freshwater vertebrate (Heteropneustes fossilis) and invertebrate (Branchiura sowerbyi), exposed to zinc oxide nanoparticles (nZnO): A General Unified Threshold model of Survival (GUTS)

The toxic effects of Zinc oxide nanoparticles (nZnO) on Branchiura sowerbyi and Heteropneustes fossilis, was assessed in a 96-hour acute exposure regime using behavioral (including loss-of balance and clumping tendencies) and physiological (mucus secretion and oxygen consumption) endpoints. While the relationship between behavioral, physiological biomarkers, and exposure concentrations was assessed using correlation analysis, nZnO toxicity was further predicted using the General Unified Threshold model for Survival (GUTS). The time-dependent lethal limits for acute nZnO toxicity (LC50) on B. sowerbyi were estimated to be 0.668, 0.588, 0.448, and 0.400 mg/l, respectively, at 24, 48, 72, and 96 h whereas for H. fossilis the LC50 values are 0.954, 0.905, 0.874 and 0.838 mg/l. Threshold effect values i.e., LOEC (Lowest Observed Effect Concentration), NOEC (No Observed Effect Concentration), and MATC (Maximum Acceptable Toxicant Concentration) threshold effect values at 96 h were higher for fish compared to the oligochaete. For B. sowerbyi, the GUTS-SD (stochastic death) model is a better predictor of nanoparticle exposure effects compared to the GUTS-IT (individual tolerance) model, however in the case of H. fossilis, the reverse pattern was observed. Oxygen consumption rate was negatively correlated to mortality under acute exposure duration. The strong negative correlation between mortality and oxygen consumption strongly suggests a metabolic-toxicity pathway for nZnO exposure effects. The higher toxicity threshold values i.e., LOEC, NOEC, and MATC for fish compared to the oligochaete invertebrate indicates greater risks for invertebrates compared to vertebrates, with resultant implications for local habitat trophic relationships.

Responses of Freshwater Planarian Girardia tigrina to Fipronil-Based Insecticide: Survival, Behavioral and Physiological Endpoints

Fipronil is a pyrazole insecticide used to control undesirable insect populations. Due to its large-scale application, there is the potential for surface waters’ contamination, with toxic action for non-target organisms, and consequent impacts on aquatic ecosystems. Planarians are potential non-target aquatic invertebrates to these insecticides. They are widespread in tropical freshwaters and have been proposed as good candidates to assess the toxic effects of freshwater systems contaminated by insecticides. Thus, the present study aims to evaluate the sublethal concentrations of a fipronil-based insecticide that may affect the planarian physiology. After chronic exposure to Regent 800 WG®, a significant decrease in locomotor velocity (LOEC—6.25 mg·L−1), regeneration of the auricles and photoreceptors (LOEC—3.13 mg·L−1), and reproduction (fecundity—LOEC 12.5 mg·L−1) were observed. The results of our study demonstrate that long-term exposure to a pyrazole insecticide can compromise non-target aquatic invertebrates while reinforcing the need for a better investigation of complementary parameters (such as behavior, regeneration, and reproduction) for a more accurate risk assessment of commercial pesticide toxicity in freshwater systems.

Evaluation of Locomotion Complexity in Zebrafish after Exposure to Twenty Antibiotics by Fractal Dimension and Entropy Analysis.

Antibiotics are extensively used in aquaculture to prevent bacterial infection and the spread of diseases. Some antibiotics have a relatively longer half-life in water and may induce some adverse effects on the targeted fish species. This study analyzed the potential adverse effects of antibiotics in zebrafish at the behavioral level by a phenomic approach. We conducted three-dimensional (3D) locomotion tracking for adult zebrafish after acute exposure to twenty different antibiotics at a concentration of 100 ppb for 10 days. Their locomotor complexity was analyzed and compared by fractal dimension and permutation entropy analysis. The dimensionality reduction method was performed by combining the data gathered from behavioral endpoints alteration. Principal component and hierarchical analysis conclude that three antibiotics: amoxicillin, trimethoprim, and tylosin, displayed unique characteristics. The effects of these three antibiotics at lower concentrations (1 and 10 ppb) were observed in a follow-up study. Based on the results, these antibiotics can trigger several behavioral alterations in adult zebrafish, even in low doses. Significant changes in locomotor behavioral activity, such as total distance activity, average speed, rapid movement time, angular velocity, time in top/bottom duration, and meandering movement are highly related to neurological motor impairments, anxiety levels, and stress responses were observed. This study provides evidence based on an in vivo experiment to support the idea that the usage of some antibiotics should be carefully addressed since they can induce a significant effect of behavioral alterations in fish.

Identifying Behavioral Response Profiles of Two Common Larval Fish Models to a Salinity Gradient.

Salinization of aquatic systems is an emerging global issue projected to increase in magnitude, frequency, and duration with climate change and landscape modifications. To consider influences of salinity on locomotor activity of common fish models, we examined behavioral response profiles of two species, zebrafish (Danio rerio) and fathead minnow (Pimephales promelas), across a gradient of sodium chloride. Following each experiment, behavior was recorded with automated tracking software and then behavioral response variables, including locomotor (e.g., distance traveled, number of movements, duration of movements) and photolocomotor changes, were examined at several speed thresholds (bursting, cruising, freezing) to identify potential salinity responses. Zebrafish responses were significantly (p < 0.05) reduced at the highest treatment level (5.78g/L) for multiple behavioral endpoints during both dark and light phases; however, fathead minnow responses were more variable and not consistently concentration dependent. Future efforts are needed to understand behavioral response profiles in combination with anthropogenic contaminants and natural toxins across the freshwater to marine continuum, considering salinization of inland waters, sea level rise, and transport of anthropogenic contaminants and algal toxins from inland waters to coastal systems.

Establishing a High-Throughput Locomotion Tracking Method for Multiple Biological Assessments in Tetrahymena.

Protozoa are eukaryotic, unicellular microorganisms that have an important ecological role, are easy to handle, and grow rapidly, which makes them suitable for ecotoxicity assessment. Previous methods for locomotion tracking in protozoa are largely based on software with the drawback of high cost and/or low operation throughput. This study aimed to develop an automated pipeline to measure the locomotion activity of the ciliated protozoan Tetrahymena thermophila using a machine learning-based software, TRex, to conduct tracking. Behavioral endpoints, including the total distance, velocity, burst movement, angular velocity, meandering, and rotation movement, were derived from the coordinates of individual cells. To validate the utility, we measured the locomotor activity in either the knockout mutant of the dynein subunit DYH7 or under starvation. Significant reduction of locomotion and alteration of behavior was detected in either the dynein mutant or in the starvation condition. We also analyzed how Tetrahymena locomotion was affected by the exposure to copper sulfate and showed that our method indeed can be used to conduct a toxicity assessment in a high-throughput manner. Finally, we performed a principal component analysis and hierarchy clustering to demonstrate that our analysis could potentially differentiate altered behaviors affected by different factors. Taken together, this study offers a robust methodology for Tetrahymena locomotion tracking in a high-throughput manner for the first time.

Assessing the importance of sex in a hippocampus-dependent behavioral test battery in C57BL/6NTac mice.

Inclusion of male and female subjects in behavioral neuroscience research requires a concerted effort to characterize sex differences in standardized behavioral assays. Sex differences in hippocampus-dependent assays have been widely reported but are still poorly characterized. In the present study, we conducted a parametric analysis of spontaneous alternation, object recognition, and fear conditioning in a commonly used control strain, C57BL/6NTac. Our findings show largely similar performance between males and females across the majority of behavioral end points. However, we identified an important difference in nonassociative fear sensitization, whereby females showed an enhanced fear response to the 75-dB tone that is used as the conditional stimulus. In addition, we observed an impairment in object location performance in females that was ameliorated by more extensive habituation to handling. Together, these findings argue that sex differences in nonassociative fear responses to both novel auditory cues and novel objects need to be considered when designing and interpreting cognitive assays in C57BL/6 mice. Furthermore, this elevated fear sensitization could serve as a novel approach to model the increased incidence of anxiety disorders in women.