Behavioral Endpoints Research Articles

Evaluation of Tacrolimus' Adverse Effects on Zebrafish in Larval and Adult Stages by Using Multiple Physiological and Behavioral Endpoints.

Tacrolimus (FK506) is a common immunosuppressant that is used in organ transplantation. However, despite its importance in medical applications, it is prone to adverse side effects. While some studies have demonstrated its toxicities to humans and various animal models, very few studies have addressed this issue in aquatic organisms, especially zebrafish. Here, we assessed the adverse effects of acute and chronic exposure to tacrolimus in relatively low doses in zebrafish in both larval and adult stages, respectively. Based on the results, although tacrolimus did not cause any cardiotoxicity and respiratory toxicity toward zebrafish larvae, it affected their locomotor activity performance in light-dark locomotion tests. Meanwhile, tacrolimus was also found to slightly affect the behavior performance, shoaling formation, circadian rhythm locomotor activity, and color preference of adult zebrafish in a dose-dependent manner. In addition, alterations in the cognitive performance of the fish were also displayed by the treated fish, indicated by a loss of short-term memory. To help elucidate the toxicity mechanism of tacrolimus, molecular docking was conducted to calculate the strength of the binding interaction between tacrolimus to human FKBP12. The results showed a relatively normal binding affinity, indicating that this interaction might only partly contribute to the observed alterations. Nevertheless, the current research could help clinicians and researchers to further understand the toxicology of tacrolimus, especially to zebrafish, thus highlighting the importance of considering the toxicity of tacrolimus prior to its usage.

Behavioral effects of 6-hydroxydopamine-induced damage to nigro-striatal pathway and Locus coeruleus as a rodent model of Parkinson’s disease

Parkinson's disease (PD) is a chronic and progressive neurodegenerative disorder characterized by the loss of dopaminergic neurons in the Substantia nigra pars compacta (SNpc), which leads to motor and non-motor symptoms (NMS). NMS can appear many years before the classical motor symptoms and are associated with the neurodegeneration of several nuclei; in this work, we highlight the neurodegeneration of Locus coeruleus (LC) in PD. The aim was to investigate the effects of depleting SNpc and LC catecholaminergic neurons on behavioral and neurobiological endpoints. Here we used 6-hydroxydopamine (6-OHDA) in order to induced neurotoxic damage in three independent experimental groups: SNpc lesion group, which 6-OHDA was injected into CPu (CPu-6-OHDA), LC lesion group, which 6-OHDA was injected directly on LC to selectively caused a damage on this nucleus (LC-6-OHDA), and the combined SNpc and LC lesion group (CL-6-OHDA). Next, the behavioral studies were performed using the Morris water maze (MWM), open field (OF), and elevated plus maze (EPM). After stereotaxic surgeries, the animals showed a loss of 67% and 77% of Tyrosine hydroxylase (TH) reactive neurons in the SNpc and LC, respectively. The behavioral analysis showed the anxiety-like behavior in CL-6-OHDA group in the EPM test; in the MWM test, the combined lesions (CL-6-OHDA) showed an impairment in memory acquisition and spatial memory; and no changes were observed in locomotor activity in all the tests. Furthermore, our investigation demonstrating the effects of depleting SN and LC catecholaminergic neurons on behavioral and neurobiological parameters. All these data together lead us to believe that a bilateral PD model including a LC bilateral degeneration is potentially a more accurate model to evaluate the NMS in the pathological development of the disease in rodents.

Neuronal Excitability in the Medial Habenula and Ventral Tegmental Area Is Differentially Modulated by Nicotine Dosage and Menthol in a Sex-Specific Manner.

The medial habenula (MHb) has been identified as the limiting factor for nicotine intake and facilitating nicotine withdrawal. However, few studies have assessed MHb neuronal excitability in response to nicotine, and, currently, a gap in knowledge is present for finding behavioral correlates to neuronal excitability in the region. Moreover, no study to date has evaluated sex or nicotine dosage as factors of excitability in the MHb. Here, we utilized an e-vape self-administration (EVSA) model to determine differences between sexes with different nicotine dosages ± menthol. Following this paradigm, we employed patch-clamp electrophysiology to assess key metrics of MHb neuronal excitability in relation to behavioral endpoints. We observed female mice self-administered significantly more than males, regardless of dosage. We also observed a direct correlation between self-administration behavior and MHb excitability with low-dose nicotine + menthol in males. Conversely, a high dose of nicotine ± menthol yields an inverse correlation between excitability and self-administration behavior in males only. In addition, intrinsic excitability in the ventral tegmental area (VTA) does not track with the amount of nicotine self-administered. Rather, they correlate to the active/inactive discrimination of mice. Using fast-scan cyclic voltammetry, we also observed that dopamine release dynamics are linked to reinforcement-related behavior in males and motivation-related behaviors in females. These results point to a sex-specific difference in the activity of the MHb and VTA leading to distinct differences in self-administration behavior. His could lend evidence to clinical observations of smoking and nicotine-use behavior differing between males and females.

Naphthenic acid fraction compounds, produced by the extraction of bitumen from oil sands, alter survival and behaviour of juvenile yellow perch (Perca flavescens)

We evaluated whether naphthenic acid fraction compounds (NAFCs) extracted from oil sand tailings adversely affect fish survival and behaviour. Following a before–after-control-impact design, we housed wild-caught juvenile yellow perch ( Perca flavescens) in outdoor mesocosms to assess survival and behaviour under baseline conditions, then exposed fish to one of three treatments: negative control, 2 mg/L NAFC, or 15 mg/L NAFC. We performed behavioural assays (no-stimulus activity, food stimulus, and predator stimulus using a model bird) and assessed a comprehensive suite of endpoints (equilibrium losses, activity, shoaling, burst swimming, freezing, and space use). We found that exposure to 15 mg/L NAFCs substantially reduced fish survival and impaired fish equilibrium in all three behavioural tests. Furthermore, exposure to NAFCs impaired anti-predator behaviour: while the activity of control fish increased by two-fold in response to a predator stimulus, fish exposed to 2 or 15 mg/L NAFC did not change their activity levels after stimulation. No significant changes were observed in other behavioural endpoints. Overall, our findings suggest that a week-long exposure to NAFCs at concentrations commonly found in tailings ponds, constructed wetlands, and other mining-impacted waters may affect multiple facets of fish behaviour that could ultimately lead to reduced fitness in fish populations.

Prepubescent Electronic Cigarette Exposure Affects Sexual Motivation and Puberty in Female But Not Male Long-Evans Rats.

A method for delivering vaporized nicotine to animals has been developed using e-cigarette devices. The present experiment was designed to measure the effects of e-cigarette nicotine on pubertal onset and development of reproductive behavior in female and male Long-Evans rats. Rats received daily 10-min sessions of electronic-cigarette vaporized nicotine (5% Virginia Tobacco JUUL Pods) or room air in a whole-body exposure chamber (postnatal day 28-31). Pubertal onset was monitored daily (ie, vaginal opening in females, preputial separation in males). Two weeks later, rats were tested for sexual motivation using the partner-preference paradigm, whereby subjects were given the opportunity to approach either a sexual partner or a same-sex social partner. Four weeks later, partner preference was assessed again, 10 min after rats were re-exposed to their same prepubertal treatment. We found that prepubescent electronic-cigarette vaporized nicotine disrupted puberty and sexual motivation in female but not male rats. In vaped females, vaginal opening was delayed and less time was spent with the male stimulus compared to room-air controls. In contrast, no effect of e-cigarette vapor was observed on pubertal onset or on any measures of sexual behavior in male rats. No effects were observed in either female or male rats on the second partner-preference test. Prepubescent vaporized nicotine affected the development of reproductive physiology and behavior in female rats but not in male rats, whereas an additional acute exposure to nicotine vapor had no effect in either female or male adult rats. Given the prevalence of increasingly younger users, more animal research is needed to explore the effects of e-cigarette smoking on multiple developmental systems including reproductive physiology and behavior. This model could be useful in exploring multiple behavioral and physiological endpoints in both sexes. Adjustments to the duration of exposure and control conditions will be necessary for future experiments to best model human use.

Safe discontinuation of psychotropic drugs in older people? : New evidence and practical approach

Many older patients are permanently prescribed one or more psychotropic drugs for treatment of symptoms, such as behavioral and psychological symptoms in dementia, depressive symptoms, anxiety, and insomnia. They therefore contribute to the risk of polypharmacy. Recently, deprescribing studies have been published in order to clarify if inadequate medications can be safely discontinued. This mini-review summarizes the study results and derives practical recommendations for routine use. A literature search was carried out in PubMed for clinical studies on deprescribing in association with psychotropic substances. After removal of duplications, 12heterogeneous clinical studies were identified and reduction of psychotropic substances could be successfully achieved in 8studies. In four of these studies psychological, behavioral and functional endpoints were reported. Criteria for successful deprescribing of sedatives were in particular motivation, information and sufficient cooperation of the patients and for antipsychotic drugs in people with dementia, the sustainable establishment of nonpharmaceutical treatment strategies. Deprescribing was not attempted in cases of ahistory of severe chronic mental illness and in cases of severe behavioral symptoms in dementia. Evidence for antidepressants was not sufficient to extract practical recommendations. Safe deprescribing of antipsychotic drugs in patients with dementia is justified if non-pharmacological treatment options are sustainably implemented, and for sedative drugs in well-informed, highly motivated and cooperative patients.

Repeated Activation of Pyramidal Neurons in the Prefrontal Cortex Alters Microglial Phenotype in Male Mice.

Aberrant neuronal activity in the cortex alters microglia phenotype and function in several contexts, including chronic psychologic stress and neurodegenerative disease. Recent findings even suggest that heightened levels of neuronal activity spur microglia to phagocytose synapses, with potential impacts on cognition and behavior. Thus, the present studies were designed to determine if activation of neurons alone-independent of disease or dysfunction-is sufficient to alter microglial phenotype in the medial prefrontal cortex (mPFC), a brain region critical in emotion regulation and cognition. In these studies, we used both an adeno-associated virus-mediated and Cre-dependent chemogenetic [designer receptors exclusively activated by designer drugs (DREADD)] approach to repeatedly activate excitatory pyramidal neurons (CaMKIIa+) neurons in the mPFC. Various molecular, cytometric, and behavioral endpoints were examined. Recurrent DREADD-induced neuronal activation led to pronounced changes in microglial density, clustering, and morphology in the mPFC and increased microglia-specific transcripts implicated in synaptic pruning (e.g., Csf1r, Cd11b). Further analyses revealed that the magnitude of DREADD-induced neuronal activation was significantly correlated with measures of microglial morphology in the mPFC. These alterations in microglial phenotype coincided with an increase in microglial lysosome volume in the mPFC and selective deficits in working memory function. Altogether, these findings indicate that repeated neuronal activation alone is sufficient to drive changes in microglia phenotype and function in the mPFC. Future studies using optogenetic and chemogenetic approaches to manipulate neural circuits need to consider microglial and other nonneuronal contributions to physiologic and behavioral outcomes. SIGNIFICANCE STATEMENT: Microglia are highly attuned to fluctuations in neuronal activity. Here we show that repeated activation of pyramidal neurons in the prefrontal cortex induces broad changes in microglia phenotype; this includes upregulation of pathways associated with microglial proliferation, microglia-neuron interactions, and lysosome induction. Our findings suggest that studies using chemogenetic or optogenetic approaches to manipulate neural circuits should be mindful of indirect effects on nonneuronal cells and their potential contribution to measured outcomes.

Assessing the disparity: comparative toxicity of Copper in zebrafish larvae exposes alarming consequences of permissible concentrations in Brazil

ABSTRACT Copper (Cu) is a naturally occurring metal with essential micronutrient properties. However, this metal might also pose increased adverse environmental and health risks due to industrial and agricultural activities. In Brazil, the maximum allowable concentration of Cu in drinking water is 2 mg/L. Despite this standard, the impact of such concentrations on aquatic organisms remains unexplored. This study aimed to evaluate the toxicity of CuSO4 using larval zebrafish at environmentally relevant concentrations. Zebrafish (Danio rerio) larvae at 72 hr post-fertilization (hpf) were exposed to nominal CuSO4 concentrations ranging from 0.16 to 48 mg/L to determine the median lethal concentration (LC50), established at 8.4 mg/L. Subsequently, non-lethal concentrations of 0.16, 0.32, or 1.6 mg/L were selected for assessing CuSO4 -induced toxicity. Morphological parameters, including body length, yolk sac area, and swim bladder area, were adversely affected by CuSO4 exposure, particularly at 1.6 mg/L (3.31 mm ±0.1, 0.192 mm2 ±0.01, and 0.01 mm2 ±0.05, respectively). In contrast, the control group exhibited values of 3.62 mm ±0.09, 0.136 mm2 ±0.013, and 0.3 mm2 ±0.06, respectively. Behavioral assays demonstrated impairments in escape response and swimming capacity, accompanied by increased levels of reactive oxygen species (ROS) and lipid peroxidation. In addition, decreased levels of non-protein thiols and reduced cellular viability were noted. Data demonstrated that exposure to CuSO4 at similar concentrations as those permitted in Brazil for Cu adversely altered morphological, biochemical, and behavioral endpoints in zebrafish larvae. This study suggests that the permissible Cu concentrations in Brazil need to be reevaluated, given the potential enhanced adverse health risks of exposure to environmental metal contamination.

Deleterious effects of benzotriazoles on zebrafish development and neurotransmission: 5-Chloro-benzotriazole versus 1H-benzotriazole

Benzotriazoles are heterocyclic compounds typically presenting a benzene ring fused with a triazole molecule. The industry uses these compounds as anti-corrosion agents and recently, they have been employed in the pharmaceutical industry and in detergent formulations. Benzotriazoles persist in the environment, and water treatment plants cannot degrade them completely. Consequently, these compounds have been detected in rivers, lakes, and drinking water, which makes assessing their safety for the human and aquatic animal populations crucial. Here, we have evaluated and compared how exposure to 1H-benzotriazole or 5-chloro-benzotriazole affect the zebrafish embryo-larval stages. We have determined the acute toxicity, morphometric alterations, and acetylcholinesterase activity on zebrafish embryos, as well as behavioral endpoints using the tail coiling assay. The estimated LC50 of 5-chloro-benzotriazole was 19 mg/L, whereas 1H-benzotriazole caused no mortality. The zebrafish embryos exposed to 20 and 25 mg/L 5-chloro-benzotriazole had decreased hatching rate and exhibited pericardial and yolk sac edemas. Furthermore, the embryo length and eye area were decreased, in contrast with an increased yolk sac after exposure to 20 mg/L 5-chloro-benzotriazole. In turn, 1H-benzotriazole also decreased the eye area of zebrafish embryos, but no other significant morphological alterations were observed. The tail coiling assay showed that the zebrafish embryos increased the percentage of time moving and the number of embryonic movements per minute after exposure to 1H-benzotriazole (15 mg/L) or 5-chloro-benzotriazole (20 and 25 mg/L), indicating that these compounds were potentially neurotoxic. However, acetylcholinesterase activity was not significantly altered in embryos exposed to 1H-benzotriazole, but significantly decreased when exposed to 0.05 mg/L 5-chloro benzotriazole confirming its neurotoxicity at a much lower concentration. Our findings showed that 5-chloro-benzotriazole seems to induce more harmful alterations to zebrafish embryos than 1H-benzotriazole. Nevertheless, 1H-benzotriazole seems to induce a direct effect on eye development for concentrations lower than the ones of 5-chloro-benzotriazole affecting zebrafish embryos.

Imiquimod-induced pruritus in female wild-type and knockin Wistar rats: underscoring behavioral scratching in a rat model for antipruritic treatments

ObjectivesAnimal models of skin disease are used to evaluate therapeutics to alleviate disease. One common clinical dermatological complaint is pruritus (itch), but there is a lack of standardization in the characterization of pre-clinical models and scratching behavior, a key itch endpoint, is often neglected. One such model is the widely used imiquimod (IMQ) mouse model of psoriasis. However, it lacks characterized behavioral attributes like scratching, nor has widely expanded to other species like rats.Given these important attributes, this study was designed to broaden the characterization beyond the expected IMQ-induced psoriasis-like skin inflammatory skin changes and to validate the role of a potential therapeutic agent for pruritus in our genetic rat model. The study included female Wistar rats and genetically modified knockin (humanized proteinase-activated receptor 2 (F2RL1) female rats, with the widely used C57BL/6 J mice as a methodology control for typical IMQ dosing.ResultsWe demonstrate that the IMQ model can be reproduced in rats, including their genetically modified derivatives, and how scratching can be used as a key behavioral endpoint. We systemically delivered an anti-PAR2 antibody (P24E1102) which reversed scratching bouts—validating this behavioral methodology and have shown its feasibility and value in identifying effective antipruritic drugs.

Toxicity of environmentally relevant concentration of PFAS chemicals in Lumbriculus variegatus (Oligochaeta, Lumbriculidae) – A multi-bioindicator study

PFAS, or per- and polyfluoroalkyl substances, are a family of man-made chemicals found in a variety of products from non-stick cookware and food wrappers to firefighting foams. PFAS are persistent and widely distributed in the environment, including aquatic environments. In this study we examined the impact of PFAS chemicals on the physiological and behavioral endpoints of Lumbriculus variegatus (i.e., blackworms). Lumbriculus variegatus is a species of freshwater annelid worm that plays key roles in shallow freshwater ecosystems. At an environmentally relevant concentration of 1 μg/L, 12-day aqueous exposure to long chain PFAS, including PFOA, PFOS and PFDA, each markedly slowed the pulse rate of the dorsal blood vessel in L. variegatus, indicating a suppressive effect on blood circulation. The mean pulse rate was reduced from 9.6 beats/minute to 6.2 and 7.0 beats/min in PFOA and PFOS, respectively (P < 0.0001). Further, PFOA, PFOS and PFDA reduced the escape responsiveness of L. variegatus to physical stimulation. The percentage of worms showing normal escape behavior was reduced from 99.0% in control to 90.6% in the PFOS exposed group (P < 0.01). In a chronic (4 week) growth study, exposure to overlying water and sediment spiked with PFOA, PFOS or PFDA reduced the total biomass and the number of worms, indicating a suppressive effect on worm population growth. For instance, PFOA and PFDA reduced the total dry biomass by 26.3% and 28.5%, respectively, compared to the control (P < 0.05). The impact of PFAS on blackworm physiology is accompanied by an increase in lipid peroxidation. The level of malondialdehyde (MDA), an indicator of lipid peroxidation, and catalase, a major antioxidant enzyme, were markedly increased in PFOA, PFOS and PFDA exposed groups. Interestingly, exposure to PFHxA, a short chain PFAS, had no detectable effect on any of the measured endpoints. Our results demonstrate that L. variegatus is highly sensitive to the toxic impact of long chain PFAS chemicals as measured by multiple endpoints including blood circulation, behavior, and population growth. Such toxicity may have a detrimental impact on L. variegatus and the freshwater ecosystems where it resides.

Evidence for neurotoxicity and oxidative stress in zebrafish embryos/larvae treated with HFPO-DA ammonium salt (GenX)

"GenX" [ammonium perfluoro (2-methyl-3-oxahexanoate] was developed as a replacement chemical for toxic perfluorinated compounds to be used in product manufacturing. Here, we assessed developmental, mitochondrial, and behavioral toxicity endpoints in zebrafish embryos/larvae exposed to GenX. GenX exerted low toxicity to zebrafish embryos/larvae up to 20 mg/L. GenX did not affect mitochondrial oxidative phosphorylation nor ATP levels. ROS levels were reduced in larvae fish exposed to 10 and 100 µg/L, indicative of an antioxidant defense; however, ROS levels were elevated in fish exposed to 1000 µg/L. Increased expression of cox1 and sod2 in GenX exposed 7-day larvae was noted. GenX (0.1 or 1 µg/L) altered transcripts associated with neurotoxicity (elavl3, gfap, gap43, manf, and tubb). Locomotor activity of larvae was reduced by 100 µg/L GenX, but only in light periods. Perturbations of anxiety-related behaviors in larvae were not observed with GenX exposure. These data inform risk assessments for long-lived perfluorinated chemicals of concern.

Uncovering the common factors of chemical exposure and behavior: Evaluating behavioral effects across a testing battery using factor analysis

Although specific environmental chemical exposures, including flame retardants, are known risk factors for neurodevelopmental disorders (NDDs), direct experimental evidence linking specific chemicals to NDDs is limited. Studies focusing on the mechanisms by which the social processing systems are vulnerable to chemical exposure are underrepresented in the literature, even though social impairments are defining characteristics of many NDDs. We have repeatedly demonstrated that exposure to Firemaster 550 (FM 550), a prevalent flame retardant mixture used in foam-based furniture and infant products, can adversely impact a variety of behavioral endpoints. Our recent work in prairie voles (Microtus ochrogaster), a prosocial animal model, demonstrated that perinatal exposure to FM 550 sex specifically impacts socioemotional behavior. Here, we utilized a factor analysis approach on a battery of behavioral data from our prior study to extract underlying factors that potentially explain patterns within the FM 550 behavior data. This approach identified which aspects of the behavioral battery are most robust and informative, an outcome critical for future study designs. Pearson’s correlation identified behavioral endpoints associated with distance and stranger interactions that were highly correlated across 5 behavioral tests. Using these behavioral endpoints, exploratory factor analysis (EFA) and confirmatory factor analysis (CFA) extracted 2 factors that could explain the data: Activity (distance traveled endpoints) and Sociability (time spent with a novel conspecific). Exposure to FM 550 significantly decreased Activity and decreased Sociability. This factor analysis approach to behavioral data offers the advantages of modeling numerous measured variables and simplifying the data set by presenting the data in terms of common, overarching factors in terms of behavioral function.

Avoidance behavior of Hyalella azteca in response to three common-use insecticides

Non-target organisms in aquatic environments may experience lethal or sublethal effects following exposure to contaminants. Most protocols and regulations, however, are designed to provide protection from lethal effects and are thus based on conventional estimates of population lethality. The relative lack of reliable behavioral endpoints makes it challenging to implement regulations that are similarly protective against sublethal toxicity. The objective of this study was to quantify the avoidance behavior of Hyalella azteca when exposed to three insecticides—bifenthrin (B), chlorpyrifos (C), and permethrin (P)—at a range of estimated lethal concentrations. A two-choice behavioral arena was used for each chemical to quantify H. azteca activity and time spent in either uncontaminated sediment or sediment spiked at concentrations reflecting estimated 48-h lethal concentrations (LC50, LC25, and LC10). For all three insecticides, naïve H. azteca demonstrated a preference for the uncontaminated sediment over the contaminated sediment at the LC50 (B: 312 ng/gOC; C: 1265 ng/gOC; P: 5042 ng/gOC) and LC25 (B: 230 ng/gOC; C: 859 ng/gOC; P: 3817 ng/gOC), spending significantly more time in the uncontaminated side of the arena. H. azteca did not avoid sediment at LC10 (B: 204 ng/gOC; C: 609 ng/gOC; P: 1515 ng/gOC) levels, indicating the existence of a potential threshold of detection. Despite the lack of substrate preference at this exposure level, H. azteca were nevertheless more active (i.e., increased zone-switching) when exposed to bifenthrin at the LC10, suggesting a possible irritation response (e.g., movement after exposure) to this chemical. Our results provide evidence that H. azteca exhibit innate avoidance responses to sediments contaminated with common insecticides at concentrations below those represented by traditional toxicological endpoints (e.g., LC50). The sensitivity and ease with which this behavioral endpoint can be assayed demonstrates the potential utility of behavioral endpoints in toxicological assessments using model organisms.

The Tetrahymena Paradigm: Genetic Insights and Ecotoxicity Assessment

Tetrahymena, a ciliated protozoan, thrives in freshwater habitats across diverse climates. Its importance as a genetic model organism lies in its cellular complexity, with cells comparable to human cells and a genome size between yeast and human. Tetrahymena’s germline genome undergoes unique chromosome breakage and DNA elimination, shedding light on genome rearrangement mechanisms. In 2020, the macronuclear genome was fully sequenced, offering improved insights into chromosome structure and gene models. Tetrahymena’s molecular tools enable gene characterization, knockouts, and overexpression. Beyond genetics, Tetrahymena serves as an ecotoxicity model, particularly Tetrahymena pyriformis. Changes in its morphology, food vacuole formation, and contractile vacuole response serve as toxicity indicators. Tetrahymena’s unique nuclear dimorphism (micronucleus and macronucleus) adds depth to ecotoxicological research. Additionally, Tetrahymena’s behavioral endpoints, including chemotaxis, phagocytosis, and motility, offer sensitive toxicity markers. Researchers have developed a T-maze toxotactic assay, a quick tool for studying Tetrahymena’s behavioral responses to different chemicals. Tetrahymena’s phagocytosis and motility are influenced by exposure to toxicants, reflecting changes in ion channels. Tetrahymena’s rapid movement makes it ideal for studying motility alterations caused by toxic substances. AI-driven tools enhance Tetrahymena behavior studies, allowing for behavior classification, feature extraction, and predictive modeling. This interdisciplinary approach aids in understanding Tetrahymena’s intricate behaviors, offering potential applications in gene discovery and pharmacological interventions. In conclusion, Tetrahymena stands as an invaluable model organism for genetic and ecotoxicological research. Its advantages, including genetic tractability and behavioral sensitivity, contribute to our understanding of fundamental biological processes and chemical safety assessment, with AI playing a growing role in advancing this field.

Digital behavioural signatures reveal trans-diagnostic clusters of Schizophrenia and Alzheimer's disease patients

The current neuropsychiatric nosological categories underlie pragmatic treatment choice, regulation and clinical research but does not encompass biological rationale. However, subgroups of patients suffering from schizophrenia or Alzheimer's disease have more in common than the neuropsychiatric nature of their condition, such as the expression of social dysfunction. The PRISM project presents here initial quantitative biological insights allowing the first steps toward a novel trans-diagnostic classification of psychiatric and neurological symptomatology intended to reinvigorate drug discovery in this area. In this study, we applied spectral clustering on digital behavioural endpoints derived from passive smartphone monitoring data in a subgroup of Schizophrenia and Alzheimer's disease patients, as well as age matched healthy controls, as part of the PRISM clinical study. This analysis provided an objective social functioning characterization with three differential clusters that transcended initial diagnostic classification and was shown to be linked to quantitative neurobiological parameters assessed. This emerging quantitative framework will both offer new ways to classify individuals in biologically homogenous clusters irrespective of their initial diagnosis, and also offer insights into the pathophysiological mechanisms underlying these clusters.

A comprehensive painkillers screening by assessing zebrafish behaviors after caudal fin amputation

Recently, the usage of zebrafish for pain studies has increased in the past years, especially due to its robust pain-stimulated behaviors. Fin amputation has been demonstrated to induce a noxious response in zebrafish. However, based on the prior study, although lidocaine, the most used painkiller in zebrafish, has been shown to ameliorate amputated zebrafish behaviors, it still causes some prolonged effects. Therefore, alternative painkillers are always needed to improve the treatment quality of fin-amputated zebrafish. Here, the effects of several analgesics in recovering zebrafish behaviors post-fin amputation were evaluated. From the results, five painkillers were found to have potentially beneficial effects on amputated fish behaviors. Overall, these results aligned with their binding energy level to target proteins of COX-1 and COX-2. Later, based on their sub-chronic effects on zebrafish survivability, indomethacin, and diclofenac were further studied. This combination showed a prominent effect in recovering zebrafish behaviors when administered orally or through waterborne exposure, even with lower concentrations. Next, based on the ELISA in zebrafish brain tissue, although some changes were found in the treated group, no statistical differences were observed in most of the tested biomarkers. However, since heatmap clustering showed a similar pattern between biochemical and behavior endpoints, the minor changes in each biomarker may be sufficient in changing the fish behaviors.

Using crayfish behavior assay as a simple and sensitive model to evaluate potential adverse effects of water pollution: Emphasis on antidepressants

The freshwater crayfish, Procambarus clarkii is an excellent aquatic animal model that is highly adaptable and tolerant. P. clarkii is widely used as a toxicity model to study various pharmaceutical exposure. This animal model has complex behavioral traits and is considered sensitive to environmental changes, making it an excellent candidate to study psychoactive drugs based on a behavioral approach. However, up to now, most behavioral studies on crayfish use manual observation and scoring that require panelists. In this study, we aim to develop an automation pipeline to analyze crayfish behavior automatically. We use a deep-learning approach to label body parts in multiple crayfish, and based on the trajectory results, the intra- or inter-individual crayfish were calculated. Reliable and fast results of several behavior endpoints in multiple crayfish were retrieved. We then validated the detection performance of numerous crayfish in specific gender groups (male-male and female-female). Based on the result, the male crayfish displayed significantly higher aggression than females. We also tested the antidepressant exposure on this animal model to evaluate the psychoactive effects of this drug. As male crayfish display more distinct agonistic behavior than females, we exposed them to sertraline (SRT) 1 ppb for 7 and 14 days. It was revealed that sertraline was able to alter several behavioral endpoints in crayfish. Significant increases in extend claw ratio, total distance moved, average speed, and rapid movement were displayed in sertraline-exposed crayfish but decreased interaction time and longest interaction time. In addition, SRT 14 days exposure could atler the aggressiveness and bold behavior In the present method, DeepLabCut (DLC) has been utilized to analyze the locomotion behavior of multiple crayfish. This established method provides rapid and accurate ecotoxicity measurements using freshwater crayfish, which beneficient and applicable for environmental research.

The role of temperature on zebrafish ontogenic development and sensitivity to pharmaceuticals

This study assessed the effects of two emerging environmental pollutants caffeine (CAF) and metformin (METF) on juvenile zebrafish, raised at 22 °C or 27 ºC, after 96 h exposure. The temperature influenced the basal behaviour of unexposed juveniles, with higher swimming activity and a greater percentage of movements at the well edges observed in fish raised at 27 °C. A significant interaction between CAF and temperature was found for behavioural endpoints, but not for AChE activity, associated with neurotoxicity effects. CAF reduced swimming distance, increased erratic swimming, and inhibited AChE activity at 22 ºC, while at 27 ºC, CAF did not affect behaviour but increased AChE. METF exposure at 22 °C decreased swimming distance and increased erratic movements, but at 27 °C no effects were detected. Overall, temperature plays a more important role in the effects induced by CAF than METF.

Cardiotoxic and neurobehavioral effects of sucralose and acesulfame in Daphnia: Toward understanding ecological impacts of artificial sweeteners

Artificial sweeteners are widely used in food and pharmaceuticals, but their stability and persistence raise concerns about their impact on aquatic life. Although standard toxicity tests do not reveal lethal effects, recent studies suggest a potential neurotoxic mode of action. Using environmentally relevant concentrations, we assessed the effects of sucralose and acesulfame, common sugar substitutes, on Daphnia magna focusing on biochemical (acetylcholinesterase activity; AChE), physiological (heart rate), and behavioural (swimming) endpoints. We found dose-dependent increases in AChE and inhibitory effects on heart rate and behaviour for both substances. Moreover, acesulfame induced a biphasic response in AChE activity, inhibiting it at lower concentrations and stimulating at higher ones. For all endpoints, the EC50 values were lower for acesulfame than for sucralose. Additionally, the relationship between acetylcholinesterase and heart rate differed depending on the substance, suggesting possible differences in the mode of action between sucralose and acesulfame. All observed EC50 values were at μg/l levels, i.e., within the levels reported for wastewater, with adverse effects observed at as low as 0.1 μg acesulfame /l. Our findings emphasise the need to re-evaluate risk assessment thresholds for artificial sweeteners and provide evidence for the neurotoxic effects of artificial sweeteners in the environment, informing international regulatory standards.