Parental exposure to antidepressants has lasting effects on offspring? A case study with zebrafish.

Fish have common neurotransmitter pathways with humans, exhibiting a significant degree of conservation and homology. Thus, exposure to fluoxetine makes fish potentially susceptible to biochemical and physiological changes, similarly to what is observed in humans. Over the years, several studies demonstrated the potential effects of fluoxetine on different fish species and at different levels of biological organization. However, the effects of parental exposure to unexposed offspring remain largely unknown. The consequences of 15-day parental exposure to relevant concentrations of fluoxetine (100 and 1000 ng/L) were assessed on offspring using zebrafish as a model organism. Parental exposure resulted in offspring early hatching, non-inflation of the swimming bladder, increased malformation frequency, decreased heart rate and blood flow, and reduced growth. Additionally, a significant behavioral impairment was also found (reduced startle response, basal locomotor activity, and altered non-associative learning during early stages and a negative geotaxis and scototaxis, reduced thigmotaxis, and anti-social behavior at later life stages). These behavior alterations are consistent with decreased anxiety, a significant increase in the expression of the monoaminergic genes slc6a4a (sert), slc6a3 (dat), slc18a2 (vmat2), mao, tph1a, and th2, and altered levels of monoaminergic neurotransmitters. Alterations in behavior, expression of monoaminergic genes, and neurotransmitter levels persisted until offspring adulthood. Given the high conservation of neuronal pathways between fish and humans, data show the possibility of potential transgenerational and multigenerational effects of pharmaceuticals' exposure. These results reinforce the need for transgenerational and multigenerational studies in fish, under realistic scenarios, to provide realistic insights into the impact of these pharmaceuticals.

Cardiac and neurobehavioral impairments in three phylogenetically distant aquatic model organisms exposed to environmentally relevant concentrations of boscalid.

Boscalid (2-Chloro-N-(4'-chlorobiphenyl-2-yl) nicotinamide), a pyridine carboxamide fungicide, is an inhibitor of the complex II of the respiration chain in fungal mitochondria. As boscalid is only moderately toxic for aquatic organisms (LC50 > 1-10 mg/L), current environmental levels of this compound in aquatic ecosystems, in the range of ng/L-µg/L, are considered safe for aquatic organisms. In this study, we have exposed zebrafish (Danio rerio), Japanese medaka (Oryzias latipes) and Daphnia magna to a range of concentrations of boscalid (1-1000 µg/L) for 24 h, and the effects on heart rate (HR), basal locomotor activity (BLA), visual motor response (VMR), startle response (SR), and habituation (HB) to a series of vibrational or light stimuli have been evaluated. Moreover, changes in the profile of the main neurotransmitters have been determined. Boscalid altered HR in a concentration-dependent manner, leading to a positive or negative chronotropic effect in fish and D. magna, respectively. While boscalid decreased BLA and increased VMR in Daphnia, these behaviors were not altered in fish. For SR and HB, the response was more species- and concentration-specific, with Daphnia exhibiting the highest sensitivity. At the neurotransmission level, boscalid exposure decreased the levels of L-aspartic acid in fish larvae and increased the levels of dopaminergic metabolites in D. magna. Our study demonstrates that exposure to environmental levels of boscalid alters cardiac activity, impairs ecologically relevant behaviors, and leads to changes in different neurotransmitter systems in phylogenetically distinct vertebrate and invertebrate models. Thus, the results presented emphasize the need to review the current regulation of this fungicide.

Developmental defects in cognition, metabolic and cardiac function following maternal exposures to low environmental levels of selective serotonin re-uptake inhibitors and tributyltin in Daphnia magna.

Aquatic organisms are exposed to low concentrations of neuro-active chemicals, many of them acting also as neuroendocrine disruptors that can be hazardous during earlier embryonic stages. The present study aims to assess how exposure early in live to environmental low concentrations of two selective serotonin reuptake inhibitors (SSRIs), fluoxetine and sertraline, and tributyltin (TBT) affected cognitive, metabolic and cardiac responses in the model aquatic crustacean Daphnia magna. To that end, newly brooded females were exposed for an entire reproductive cycle (3-4 days) and the response of collected juveniles in the first, second and third consecutive broods, which were exposed, respectively, as embryos, provisioned and un-provisioned egg stages, was monitored. Pre-exposure to the selected SSRIs during embryonic and egg developmental stages altered the swimming behaviour of D. magna juveniles to light in a similar way reported elsewhere by serotonergic compounds while TBT altered cognition disrupting multiple neurological signalling routes. The studied compounds also altered body size, the amount of storage lipids in lipid droplets, heart rate, oxygen consumption rates and the transcription of related serotonergic, dopaminergic and lipid metabolic genes in new-born individuals, mostly pre-exposed during their embryonic and provisioning egg stages. The obtained cognitive, cardiac and metabolic defects in juveniles developed from exposed sensitive pre-natal stages align with the "Developmental Origins of Health and Disease (DoHAD)" paradigm.

Short-term exposure to environmental levels of nicotine and cotinine impairs visual motor response in zebrafish larvae through a similar mode of action: Exploring the potential role of zebrafish α7 nAChR.

The current view is that environmental levels of nicotine and cotinine, commonly in the ng/L range, are safe for aquatic organisms. In this study, 7 days post-fertilization zebrafish embryos have been exposed for 24 h to a range of environmental concentrations of nicotine (2.0 ng/L-2.5 µg/L) and cotinine (50 pg/L-10 µg/L), as well as to a binary mixture of these emerging pollutants. Nicotine exposure led to hyperactivity, decreased vibrational startle response and increased non-associative learning. However, the more consistent effect found for both nicotine and cotinine was a significant increase in light-off visual motor response (VMR). The effect of both pollutants on this behavior occurred through a similar mode of action, as the joint effects of the binary mixture of both chemicals were consistent with the concentration addition concept predictions. The results from docking studies suggest that the effect of nicotine and cotinine on light-off VMR could be mediated by zebrafish α7 nAChR expressed in retina. The results presented in this study emphasize the need to revisit the environmental risk assessment of chemicals including additional ecologically relevant sublethal endpoints.

Analysis of sleep/wake cycles in zebrafish larvae.

Zebrafish larvae are a model organism increasingly used in the study of the effect of neuroactive chemicals on vertebrate sleep/wake cycles. Sleep disturbances have a negative impact on mood, cognition and overall health. Here we present a protocol to assess over 24 h sleep/wake cycles in zebrafish larvae subjected to 12 h light/dark periods in 48-well plates, using video-tracking technologies. The protocol can be used to determine if the exposure to environmental pollutants or drugs can lead to sleep disturbances. The results on the effect of the tire rubber-derived 6PPD-quinone on zebrafish sleep/wake cycles presented here demonstrate the suitability of using this protocol in fish neurotoxicity studies. This protocol provides a new relevant tool to be used in the pharmacology and toxicology fields.

From dysbiosis to neuropathologies: Toxic effects of glyphosate in zebrafish.

Glyphosate, a globally prevalent herbicide known for its selective inhibition of the shikimate pathway in plants, is now implicated in physiological effects on humans and animals, probably due to its impacts in their gut microbiomes which possess the shikimate pathway. In this study, we investigate the effects of environmentally relevant concentrations of glyphosate on the gut microbiota, neurotransmitter levels, and anxiety in zebrafish. Our findings demonstrate that glyphosate exposure leads to dysbiosis in the zebrafish gut, alterations in central and peripheral serotonin levels, increased dopamine levels in the brain, and notable changes in anxiety and social behavior. While the dysbiosis can be attributed to glyphosate's antimicrobial properties, the observed effects on neurotransmitter levels leading to the reported induction of oxidative stress in the brain indicate a novel and significant mode of action for glyphosate, namely the impairment of the microbiome-gut-axis. While further investigations are necessary to determine the relevance of this mechanism in humans, our findings shed light on the potential explanation for the contradictory reports on the safety of glyphosate for consumers.

Neurotoxicity Assessment in Adult Danio rerio using a Battery of Behavioral Tests in a Single Tank.

The presence of neuropathological effects proved to be, for many years, the main endpoint for assessing the neurotoxicity of a chemical substance. However, in the last 50 years, the effects of chemicals on the behavior of model species have been actively investigated. Progressively, behavioral endpoints were incorporated into neurotoxicological screening protocols, and these functional outcomes are now routinely used to identify and determine the potential neurotoxicity of chemicals. Behavioral assays in adult zebrafish provide a standardized and reliable means to study a wide range of behaviors, including anxiety, social interaction, learning, memory, and addiction. Behavioral assays in adult zebrafish typically involve placing the fish in an experimental arena and recording and analyzing their behavior using video tracking software. Fish can be exposed to various stimuli, and their behavior can be quantified using a variety of metrics. The novel tank test is one of the most accepted and widely used tests to study anxiety-like behavior in fish. The shoaling and social preference tests are useful in studying the social behavior of zebrafish. This assay is particularly interesting since the behavior of the entire shoal is studied. These assays have proven to be highly reproducible and sensitive to pharmacological and genetic manipulations, making them valuable tools for studying the neural circuits and molecular mechanisms underlying behavior. Additionally, these assays can be used in drug screening to identify compounds that may be potential modulators of behavior. We will show in this work how to apply behavioral tools in fish neurotoxicology, analyzing the effect of methamphetamine, a recreational drug, and glyphosate, an environmental pollutant. The results demonstrate the significant contribution of behavioral assays in adult zebrafish to the understanding of the neurotoxicological effects of environmental pollutants and drugs, in addition to providing insights into the molecular mechanisms that may alter neuronal function.

Impact of environmentally relevant concentrations of fluoxetine on zebrafish larvae: From gene to behavior.

Fluoxetine is widely prescribed for the treatment of depressive states, acting at the level of the central nervous system, consequently affecting non-target organisms. This study aimed to investigate the influence of environmentally relevant fluoxetine concentrations (1-1000 ng/L) on Danio rerio development, assessing both embryotoxicity and behavior, antioxidant defense, gene expression and neurotransmitter levels at larval stage. Exposure to fluoxetine during early development was found to be able to accelerate embryo hatching in embryos exposed to 1, 10 and 100 ng/L, reduce larval size in 1000 ng/L, and increase heart rate in 10, 100 and 1000 ng/L exposed larvae. Behavioral impairments (decreased startle response and increased larvae locomotor activity) were associated with effects on monoaminergic systems, detected through the downregulation of key genes (vmat2, mao, tph1a and th2). In addition, altered levels of neurochemicals belonging to the serotonergic and dopaminergic systems (increased levels of tryptophan and norepinephrine) highlighted the sensitivity of early life stages of zebrafish to low concentrations of fluoxetine, inducing effects that may compromise larval survival. The obtained data support the necessity to test low concentrations of SSRIs in environmental risk assessment and the use of biomarkers at different levels of biological organization for a better understanding of modes of action.

First-Generation Synthetic Cathinones Produce Arrhythmia in Zebrafish Eleutheroembryos: A New Approach Methodology for New Psychoactive Substances Cardiotoxicity Evaluation.

The increasing number of new psychoactive substances (NPS) entering the illicit drug market, especially synthetic cathinones, as well as the risk of cardiovascular complications, is intensifying the need to quickly assess their cardiotoxic potential. The present study aims to evaluate the cardiovascular toxicity and lethality induced by first-generation synthetic cathinones (mephedrone, methylone, and MDPV) and more classical psychostimulants (cocaine and MDMA) in zebrafish embryos using a new approach methodology (NAM). Zebrafish embryos at 4 dpf were exposed to the test drugs for 24 h to identify drug lethality. Drug-induced effects on ventricular and atrial heart rate after 2 h exposure were evaluated, and video recordings were properly analyzed. All illicit drugs displayed similar 24 h LC50 values. Our results indicate that all drugs are able to induce bradycardia, arrhythmia, and atrial-ventricular block (AV block), signs of QT interval prolongation. However, only MDPV induced a different rhythmicity change depending on the chamber and was the most potent bradycardia and AV block-inducing drug compared to the other tested compounds. In summary, our results strongly suggest that the NAM presented in this study can be used for screening NPS for their cardiotoxic effect and especially for their ability to prolong the QT intervals.

Environmental concentrations of tire rubber-derived 6PPD-quinone alter CNS function in zebrafish larvae.

N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine quinone (6PPD-quinone) is a degradation product of 6PPD, an antioxidant widely used in rubber tires. 6PPD-quinone enters aquatic ecosystems through urban stormwater runoff and has been identified as the chemical behind the urban runoff mortality syndrome in coho salmon. However, the available data suggest that the acute effects of 6PPD-quinone are restricted to a few salmonid species and that the environmental levels of this chemical should be safe for most fish. In this study, larvae of a "tolerant" fish species, Danio rerio, were exposed to three environmental concentrations of 6PPD-quinone for only 24 h, and the effects on exploratory behavior, escape response, nonassociative learning (habituation), neurotransmitter profile, wake/sleep cycle, circadian rhythm, heart rate and oxygen consumption rate were analyzed. Exposure to the two lowest concentrations of 6PPD-quinone resulted in altered exploratory behavior and habituation, an effect consistent with some of the observed changes in the neurotransmitter profile, including increased levels of acetylcholine, norepinephrine, epinephrine and serotonin. Moreover, exposure to the highest concentration tested altered the wake/sleep cycle and the expression of per1a, per3 and cry3a, circadian clock genes involved in the negative feedback loop. Finally, a positive chronotropic effect of 6PPD-quinone was observed in the hearts of the exposed fish. The results of this study emphasize the need for further studies analyzing the effects of 6PPD-quinone in "tolerant" fish species.

Heart rate and behavioral responses in three phylogenetically distant aquatic model organisms exposed to environmental concentrations of carbaryl and fenitrothion.

Carbaryl and fenitrothion are two insecticides sharing a common mode of action, the inhibition of the acetylcholinesterase (AChE) activity. Their use is now regulated or banned in different countries, and the environmental levels of both compounds in aquatic ecosystems have decreased to the range of pg/L to ng/L. As these concentrations are below the non-observed-adverse-effect-concentrations (NOAEC) for AChE inhibition reported for both compounds in aquatic organisms, there is a general agreement that the current levels of these two chemicals are safe for aquatic organisms. In this study we have exposed zebrafish, Japanese medaka and Daphnia magna to concentrations of carbaryl and fenitrothion under their NOAECs for 24-h, and the effects on heart rate (HR), basal locomotor activity (BLA), visual motor response (VMR), startle response (SR) and its habituation have been evaluated. Both pesticides increased the HR in the three selected model organisms, although the intensity of this effect was chemical-, concentration- and organism-dependent. The exposure to both pesticides also led to a decrease in BLA and an increase in VMR in all three species, although this effect was only significant in zebrafish larvae. For SR and its habituation, the response profile was more species- and concentration-specific. The results presented in this manuscript demonstrate that concentrations of carbaryl and fenitrothion well below their respective NOAECs induce tachycardia and the impairment of ecologically relevant behaviors in phylogenetically distinct aquatic model organisms, both vertebrates and invertebrates, emphasizing the need to include this range of concentrations in the environmental risk assessment.

Developmental exposure to sertraline impaired zebrafish behavioral and neurochemical profiles.

The number of people suffering from mental health problems is rising, with anxiety and depression now the most commonly diagnosed psychiatric conditions. Selective serotonin reuptake inhibitors (SSRIs) are one of the most prescribed pharmaceuticals to treat these conditions, which has led to their common detection in many aquatic ecosystems. As the monoaminergic system shows a high degree of structural conservation across diverse animal phyla, a reasonable assumption is that the environmental levels of SSRIs in surface water can lead to adverse effects on fish and other aquatic wildlife. For instance, Sertraline (SER), a widely prescribed SSRI, has been shown to induce adverse effects in fish, albeit most of the reports used exposure concentrations exceeding those occurring in natural environments. Therefore, there is still a great lack of knowledge regarding SERs effects in fish species, especially during early life stages. This study describes the evaluation of developmental exposure of zebrafish (Danio rerio) to environmentally relevant concentrations of SER (from 0.01 to 10 µg/L), using a battery of key survival behaviors and further relating them with the expression of genes and neurochemical profiles of the monoaminergic system. We found that developmental exposure to SER did not affect embryo morphogenesis and growth. However, concentrations as low as 0.1 µg/L induced hypolocomotion and delayed learning. The observed behavioral impairment was associated with augmented serotonin levels rather than other neurochemicals and molecular markers, highlighting the relationship between serotonin signaling and behavior in zebrafish.

Caged-carvedilol as a new tool for visible-light photopharmacology of ß-adrenoceptors in native tissues.

Adrenoceptors are G protein-coupled receptors involved in a large variety of physiological processes, also under pathological conditions. This is due in large part to their ubiquitous expression in the body exerting numerous essential functions. Therefore, the possibility to control their activity with high spatial and temporal precision would constitute a valuable research tool. In this study, we present a caged version of the approved non-selective ß-adrenoceptor antagonist carvedilol, synthesized by alkylation of its secondary amine with a coumarin derivative. Introducing this photo-removable group abolished carvedilol physiological effects in cell cultures, mouse isolated perfused hearts and living zebrafish larvae. Only after visible light application, carvedilol was released and the different physiological systems were pharmacologically modulated in a similar manner as the control drug. This research provides a new photopharmacological tool for a wide range of research applications that may help in the development of future precise therapies.

Behavioral Impairment in Aquatic Organisms Exposed to Neurotoxic Pollutants.

Neuroactive chemicals are compounds that can modulate, at very low concentrations, the normal function of the central nervous systems of an organism through various primary modes of action (MoA) [...].

A Photoswitchable Ligand Targeting the ß1 -Adrenoceptor Enables Light-Control of the Cardiac Rhythm.

Catecholamine-triggered ß-adrenoceptor (ß-AR) signaling is essential for the correct functioning of the heart. Although both ß1 - and ß2 -AR subtypes are expressed in cardiomyocytes, drugs selectively targeting ß1 -AR have proven this receptor as the main target for the therapeutic effects of beta blockers in the heart. Here, we report a new strategy for the light-control of ß1 -AR activation by means of photoswitchable drugs with a high level of ß1 -/ß2 -AR selectivity. All reported molecules allow for an efficient real-time optical control of receptor function in vitro. Moreover, using confocal microscopy we demonstrate that the binding of our best hit, pAzo-2, can be reversibly photocontrolled. Strikingly, pAzo-2 also enables a dynamic cardiac rhythm management on living zebrafish larvae using light, thus highlighting the therapeutic and research potential of the developed photoswitches. Overall, this work provides the first proof of precise control of the therapeutic target ß1 -AR in native environments using light.

Environmental levels of carbaryl impair zebrafish larvae behaviour: The potential role of ADRA2B and HTR2B.

The insecticide carbaryl is commonly found in indirectly exposed freshwater ecosystems at low concentrations considered safe for fish communities. In this study, we showed that after only 24 h of exposure to environmental concentrations of carbaryl (0.066-660 ng/L), zebrafish larvae exhibit impairments in essential behaviours. Interestingly, the observed behavioural effects induced by carbaryl were acetylcholinesterase-independent. To elucidate the molecular initiating event that resulted in the observed behavioural effects, in silico predictions were followed by in vitro validation. We identified two target proteins that potentially interacted with carbaryl, the α2B adrenoceptor (ADRA2B) and the serotonin 2B receptor (HTR2B). Using a pharmacological approach, we then tested the hypothesis that carbaryl had antagonistic interactions with both receptors. Similar to yohimbine and SB204741, which are prototypic antagonists of ADRA2B and HTR2B, respectively, carbaryl increased the heart rate of zebrafish larvae. When we compared the behavioural effects of a 24-h exposure to these pharmacological antagonists with those of carbaryl, a high degree of similarity was found. These results strongly suggest that antagonism of both ADRA2B and HTR2B is the molecular initiating event that leads to adverse outcomes in zebrafish larvae that have undergone 24 h of exposure to environmentally relevant levels of carbaryl.

Low concentrations of ciprofloxacin alone and in combination with paracetamol induce oxidative stress, upregulation of apoptotic-related genes, histological alterations in the liver, and genotoxicity in Danio rerio.

Nowadays, there are countless articles about the harmful effects of paracetamol (PCM) in non-target organisms. Nonetheless, information regarding the toxicity of ciprofloxacin (CPX) and the CPX-PCM mixture is still limited. Herein, we aimed to evaluate the hepatotoxic and genotoxic effects that ciprofloxacin alone and in combination with paracetamol may induce in Danio rerio adults. For this purpose, we exposed several D. rerio adults to three environmentally relevant concentrations of PCM (0.125, 0.250, and 0.500 µg/L), CPX (0.250, 0.500, and 1 µg/L), and their mixture (0.125 + 0.250, 0.250 + 0.500, and 0.500 + 1 µg/L) for 96 h. The blood samples showed CPX alone and in combination with PCM damaged the liver function of fish by increasing the serum levels of liver enzymes alanine aminotransferase and alkaline phosphatase. Moreover, our histopathological study demonstrated liver of fish suffered several tissue alterations, such as congestion, hyperemia, infiltration, sinusoidal dilatation, macrovascular fatty degeneration, and pyknotic nuclei after exposure to CPX alone and in combination with PCM. Concerning oxidative stress biomarkers and the expression of genes, we demonstrated that CPX and its mixture, with PCM, increased the levels of antioxidant enzymes and oxidative damage biomarkers and altered the expression of Nrf1, Nrf2, BAX, and CASP3, 6, 8, and 9 in the liver of fish. Last but not least, we demonstrated CPX alone and with PCM induced DNA damage via comet assay and increased the frequency of micronuclei in a concentration-dependent manner in fish. Overall, our results let us point out CPX, even at low concentrations, induces hepatotoxic effects in fish and that its combination with PCM has a negative synergic effect in the liver of this organism.

Teratogenic effects induced by paracetamol, ciprofloxacin, and their mixture on Danio rerio embryos: Oxidative stress implications.

Even though the toxic effects of paracetamol (PCM) and ciprofloxacin (CPX) have been deeply studied in the last decades, the impact of the PCM-CPX mixture may induce in aquatic organisms is poorly known. Thus, the objective of this work was to investigate the teratogenic effects and oxidative stress that PCM, CPX, and their mixture induce in Danio rerio embryos. Moreover, we aimed to determine whether the PCM-CPX mixture induces more severe effects on the embryos than the individual drugs. For this purpose, zebrafish embryos (4 hpf) were exposed to environmentally relevant concentrations of PCM, CPX, and their mixture until 96 hpf. In addition, at 72 hpf and 96 hpf, we also evaluated the oxidative stress biomarkers (superoxide dismutase, catalase, glutathione peroxidase, lipid peroxidation, and hydroperoxides and carbonyl content) in the embryos. Our results demonstrated that PCM, CPX, and their mixture reduced the survival rate of embryos by up to 75%. In addition, both drugs, induced morphological alterations in the embryos, causing their death. The most observed malformations were: scoliosis, craniofacial malformations, hypopigmentation, growth retardation, pericardial edema. Concerning oxidative stress, our integrated biomarkers response (IBR) analysis demonstrated that PCM, CPX, and their mixture induce oxidative damage on the embryos. In conclusion, PCM, CPX, and their mixture can alter zebrafish embryonic development via an oxidative stress response.

A Zebrafish Model of Neurotoxicity by Binge-Like Methamphetamine Exposure.

Hyperthermia is a common confounding factor for assessing the neurotoxic effects of methamphetamine (METH) in mammalian models. The development of new models of methamphetamine neurotoxicity using vertebrate poikilothermic animals should allow to overcome this problem. The aim of the present study was to develop a zebrafish model of neurotoxicity by binge-like methamphetamine exposure. After an initial testing at 20 and 40 mg/L for 48 h, the later METH concentration was selected for developing the model and the effects on the brain monoaminergic profile, locomotor, anxiety-like and social behaviors as well as on the expression of key genes of the catecholaminergic system were determined. A concentration- and time-dependent decrease in the brain levels of dopamine (DA), norepinephrine (NE) and serotonin (5-HT) was found in METH-exposed fish. A significant hyperactivity was found during the first hour of exposure, followed 3 h after by a positive geotaxis and negative scototaxis in the novel tank and in the light/dark paradigm, respectively. Moreover, the behavioral phenotype in the treated fish was consistent with social isolation. At transcriptional level, th1 and slc18a2 (vmat2) exhibited a significant increase after 3 h of exposure, whereas the expression of gfap, a marker of astroglial response to neuronal injury, was strongly increased after 48 h exposure. However, no evidences of oxidative stress were found in the brain of the treated fish. Altogether, this study demonstrates the suitability of the adult zebrafish as a model of METH-induced neurotoxicity and provides more information about the biochemical and behavioral consequences of METH abuse.

Pharmacological modulation of fish-induced depth selection in D. magna: the role of cholinergic and GABAergic signalling.

Animal behaviour is closely related to individual fitness, which allows animals to choose suitable mates or avoid predation. The central nervous system regulates many aspects of animal behaviour responses. Therefore, behavioural responses can be especially sensitive to compounds with a neurodevelopmental or neurofunctional mode of action. Phototactic behavioural changes against fish in the freshwater crustacean Daphnia magna have been the subject of many ecological investigations. The aim of this study was to identify which neurotransmitter systems modulate phototactic behaviour to fish kairomones. We used a positive phototactic D. magna clone (P132,85) that shows marked negative phototactism after exposure to fish kairomones. Treatments included up to 16 known agonists and antagonists of the serotonergic, cholinergic, dopaminergic, histaminergic, glutamatergic and GABAergic systems. It was hypothesized that many neurological signalling pathways may modulate D. magna phototactic behaviour to fish kairomones. A new custom-designed device with vertically oriented chambers was used, and changes in the preferred areas (bottom, middle, and upper areas) were analysed using groups of animals after 24 h of exposure to the selected substance(s). The results indicated that agonists of the muscarinic acetylcholine and GABAA receptors and their equi-effective mixture ameliorated the negative phototactic response to fish kairomones, whereas antagonists and their mixtures increased the negative phototactism to fish kairomones. Interestingly, inhibition of the muscarinic acetylcholine receptor abolished positive phototaxis, thus inducing the phototactic response to fish kairomones. Analysis of the profile of neurotransmitters and their related metabolites showed that the D. magna behavioural responses induced by fish depend on changes in the levels of acetylcholine, dopamine and GABA.