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15 p.-7 fig. Faria, Melissa et al. ; Terrorist use of organophosphorus-based nerve agents and toxic industrial chemicals against civilian populations constitutes a real threat, as demonstrated by the terrorist attacks in Japan in the 1990 s or, even more recently, in the Syrian civil war. Thus, development of more effective countermeasures against acute organophosphorus poisoning is urgently needed. Here, we have generated and validated zebrafish models for mild, moderate and severe acute organophosphorus poisoning by exposing zebrafish larvae to different concentrations of the prototypic organophosphorus compound chlorpyrifos-oxon. Our results show that zebrafish models mimic most of the pathophysiological mechanisms behind this toxidrome in humans, including acetylcholinesterase inhibition, N-methyl-D-aspartate receptor activation, and calcium dysregulation as well as inflammatory and immune responses. The suitability of the zebrafish larvae to in vivo high-throughput screenings of small molecule libraries makes these models a valuable tool for identifying new drugs for multifunctional drug therapy against acute organophosphorus poisoning. ; This work was supported in part by the US Army ERDC-IRO (W912HZ-13-BAA-01; D.R., N.G.R., and P.J.B.) and Environmental Quality Research Program (N.G.R.), the NATO SfP project MD.SFPP 984777 (D.R., N.G.R., and P.J.B.), the National Science Foundation EPSCOR Grant EPS-0903787 (N.G.R.), the Portuguese Foundation for Science and Technology Grant SFRH/BPD/78342/2011 (Programme POPH – QREN through the Portuguese Ministry of Education and Science and the European Social Fund; M.F.), the Advanced Grant ERC-2012-AdG-320737 (D.R.) and the Spanish Government (CTM2014-51985-R; D.R.). ; Peer reviewed

In this work the effect of chlorpyrifos exposure on metabolic profiles of zebrafish muscle was evaluated by liquid chromatography coupled to high resolution mass spectrometry. Different chemometric tools based on the selection of Regions of Interest and on Multivariate Curve-Resolution-Alternating Least Squares are proposed for the analysis of the complex data sets generated in the different exposure experiments. Analysis of Variance Simultaneous Component Analysis of changes on metabolite peak profile areas showed significant chlorpyrifos concentration and exposure time-dependent changes, clearly differentiating between exposed and non-exposed samples and between short (2 h) and long exposure times (6 h or 24 h). The changes observed in the concentrations of 50 muscle metabolites are indicative of induction of oxidative stress, of a general disruption of neurotransmitter metabolism, and of muscle exhaustion. These three effects are intimately related to the toxicity of chlorpyrifos. © 2016 Elsevier Ltd ; The research leading to these results has received funding from the European Research Council under European Union's Seven Framework Programme (FP/2007-2013)/ERC Grant Agreement n.320737. ; Peer reviewed

This study examines the effects of acute pharmacological modulation of the serotonergic system over zebrafish larvae's cognitive, basic, and defense locomotor behaviors, using a medium to high throughput screening assay. Furthermore, the relationship between behavior, enzyme activity related to neurotransmitter metabolism, neurotransmitter levels, and gene expression was also determined. Modulation of larvae serotonergic system was accomplished by 24 h exposure to single and opposite pharmacodynamics co-exposure to three model psychopharmaceuticals with antagonistic and agonistic serotonin signaling properties: 2.5 mM 4-Chloro-DL-phenylalanine (PCPA) and 5 µM deprenyl and 0.5 µM fluoxetine, respectively. Similar behavioral outcome was observed for deprenyl and fluoxetine, which was reflected as hypolocomotion, decrease in larvae defensive responses, and cognitive impairment. Contrarily, PCPA induced hyperlocomotion and increase in larvae escape response. Deprenyl exposure effects were more pronounced at a lower level of organization than fluoxetine, with complete inhibition of monoamine oxidase (MAO) activity, dramatic increase of 5-HT and dopamine (DA) levels, and downregulation of serotonin synthesis and transporter genes. PCPA showed mainly effects over serotonin and dopamine's main degradation metabolites. Finally, co-exposure between agonistic and antagonist serotonin signaling drugs reviled full recovery of zebrafish impaired locomotor and defense responses, 5-HT synthesis gene expression, and partial recovery of 5-HT levels. The findings of this study suggest that zebrafish larvae can be highly sensitive and a useful vertebrate model for short-term exposure to serotonin signaling changes. ; This work was supported by the "Agencia Estatal de Investigación" from the Spanish Ministry of Science and Innovation and the IDAEA-CSIC, a Centre of Excellence Severo Ochoa (CEX2018-000794-S), through the SYNERGY Project "Environmental risk of complex mixtures of emerging persistent and mobile chemicals in aquatic ecosystems (MixPersiRisk)" financed with Severo Ochoa funds. The work was partially supported by the network of recognized research groups by the Catalan Government (2017 SGR_902). ; Peer reviewed

There is a growing interest in biological models to investigate the effect of neurotransmitter dysregulation on the structure and function of the central nervous system (CNS) at different stages of development. Zebrafish, a vertebrate model increasingly used in neurobiology and neurotoxicology, shares the common neurotransmitter systems with mammals, including glutamate, GABA, glycine, dopamine, norepinephrine, epinephrine, serotonin, acetylcholine, and histamine. In this study, we have evaluated the performance of liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the multiresidue determination of neurotransmitters and related metabolites. In a first step, ionization conditions were tested in positive electrospray mode and optimum fragmentation patterns were determined to optimize two selected reaction monitoring (SRM) transitions. Chromatographic conditions were optimized considering the chemical structure and chromatographic behavior of the analyzed compounds. The best performance was obtained with a Synergy Polar-RP column, which allowed the separation of the 38 compounds in 30 min. In addition, the performance of LC-MS/MS was studied in terms of linearity, sensitivity, intra- and inter-day precision, and overall robustness. The developed analytical method was able to quantify 27 of these neurochemicals in zebrafish chemical models for mild (P1), moderate (P2), and severe (P3) acute organophosphorus poisoning (OPP). The results show a general depression of synaptic-related neurochemicals, including the excitatory and inhibitory amino acids, as well as altered phospholipid metabolism, with specific neurochemical profiles associated to the different grades of severity. These results confirmed that the developed analytical method is a new tool for neurotoxicology research using the zebrafish model. © 2018, Springer-Verlag GmbH Germany, part of Springer Nature. ; The research leading to these results has received funding from the European Research Council under European Union's Seven Framework Programme (FP/2007e2013)/ERC Grant Agreement n.320737, the NATO SfP project MD.SFPP 984777, and the grant CTM 2014-51985 from the Spanish Ministry of Economy, Industry and Competitiveness. Moreover, Rita Bausano is acknowledged for extraction samples support during her Erasmus Traineeship stage in the Environmental Chemistry Department at IDEA-CSIC (Barcelona, Spain). ; Peer reviewed

Acute organophosphorus (OP) intoxication is a worldwide clinical and public health problem. In addition to cholinergic crisis, neurodegeneration and brain damage are hallmarks of the severe form of this toxidrome. Recently, we generated a chemical model of severe acute OP intoxication in zebrafish that is characterized by altered head morphology and brain degeneration. The pathophysiological pathways resulting in brain toxicity in this model are similar to those described in humans. The aim of this study was to assess the predictive power of this zebrafish model by testing the effect of a panel of drugs that provide protection in mammalian models. The selected drugs included "standard therapy" drugs (atropine and pralidoxime), reversible acetylcholinesterase inhibitors (huperzine A, galantamine, physostigmine and pyridostigmine), N-methyl-d-aspartate (NMDA) receptor antagonists (MK-801 and memantine), dual-function NMDA receptor and acetylcholine receptor antagonists (caramiphen and benactyzine) and anti-inflammatory drugs (dexamethasone and ibuprofen). The effects of these drugs on zebrafish survival and the prevalence of abnormal head morphology in the larvae exposed to 4 µM chlorpyrifos oxon [1 × median lethal concentration (LC50)] were determined. Moreover, the neuroprotective effects of pralidoxime, memantine, caramiphen and dexamethasone at the gross morphological level were confirmed by histopathological and transcriptional analyses. Our results demonstrated that the zebrafish model for severe acute OP intoxication has a high predictive value and can be used to identify new compounds that provide neuroprotection against severe acute OP intoxication. © 2016, The Author(s). ; This work was supported in part by the US Army ERDC-IRO (W912HZ-13-BAA-01; D.R.), the NATO SfP project MD.SFPP 984777 (D.R.), the Portuguese Foundation for Science and Technology Grant SFRH/BPD/78342/2011 (Programme POPH—QREN through the Portuguese Ministry of Education and Science and the European Social Fund; M.F.) and the Spanish Government (CTM2014-51985-R; D.R., E.P, M.F.). We also are grateful to Dr. Martí Pumarola from the Departament de Medicina i Cirurgia Animals of the UAB for their valuable comments on neuropathology. ; Peer reviewed

Occupational, accidental, or suicidal exposure to acrylamide (ACR) may result in a neurotoxic syndrome. Development of animal models of acrylamide neurotoxicity is necessary for increasing our mechanistic understanding of this syndrome and developing more effective therapies. A new model for acute ACR neurotoxicity has been recently developed in adult zebrafish. Whereas the results of the initial characterization were really promising, a further characterization is needed for testing the construct validity of the model. In this study, the presence of gait abnormalities has been investigated by using ZebraGait, software specifically designed to analyze the kinematics of fish swimming in a water tunnel. The results of the kinematic analyses demonstrated that the model exhibits mild-to-moderate gait abnormalities. Moreover, the model exhibited negative scototaxis, a result confirming a phenotype of anxiety comorbid with depression phenotype. Interestingly, depletion of the reduced glutathione levels was found in the brain without a concomitant increase in oxidative stress. Finally, hypolocomotion and positive geotaxis exhibited by this model were fully recovered 5 days after transferring the fish to clean fish-water. All this data support the validity of the ACR acute neurotoxicity model developed in adult zebrafish. ; We thank Dr. Caio Maximino, from the Universidade Federal do Sul e Sudeste do Pará (Marabá, Brazil), for his comments and suggestions on the DLT. The IRI Lab technical staff members are gratefully thanked for their valuable assistance in the design of the experimental setup for video-recording the fish swimming. This work was supported by the NATO SfP project MD. SFPP 984777 (D.R.) and the Spanish Government (CTM2017-83242-R; D.R. and J.M.P.). M.F. acknowledges financial support from the Beatriu de Pinós programme (Grant No. 2016 BP 00233) provided by the Secretariat of Universities and Research department of the Ministry for Business and Knowledge, Catalonia Government. ; Peer reviewed

Changes on an organism by the exposure to environmental stressors may be characterized by hyperspectral images (HSI), which preserve the morphology of biological samples, and suitable chemometric tools. The approach proposed allows assessing and interpreting the effect of contaminant exposure on heterogeneous biological samples monitored by HSI at specific tissue levels. In this work, the model example used consists of the study of the effect of the exposure of chlorpyrifos-oxon on zebrafish tissues. To assess this effect, unmixing of the biological sample images followed by tissue-specific classification models based on the unmixed spectral signatures is proposed. Unmixing and classification are performed by multivariate curve resolution-alternating least squares (MCR-ALS) and partial least squares-discriminant analysis (PLS-DA), respectively. Crucial aspects of the approach are: (1) the simultaneous MCR-ALS analysis of all images from 1 population to take into account biological variability and provide reliable tissue spectral signatures, and (2) the use of resolved spectral signatures from control and exposed populations obtained from resampling of pixel subsets analyzed by MCR-ALS multiset analysis as information for the tissue-specific PLS-DA classification models. Classification results diagnose the presence of a significant effect and identify the spectral regions at a tissue level responsible for the biological change. ; The research leading to these results has received funding from the European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013) / ERC Grant Agreement n. 32073 (CHEMAGEB project). The authors of this work belong to the network of recognized research groups by the Catalan government (2014 SGR 1106) and acknowledge the support of the Spanish government through project CTQ2015-66254-C2-2-P. ICFO would like to acknowledge financial support from Laserlab-Europe (EU-H2020 654148), the Spanish MINECO (Severo Ochoa grant SEV-2015-0522), Marató de TV3 (20142030), and the National Institute of Health (NIH, grant 5R21CA187890-02). The research conducted at ICFO's Super Resolution Light Microscopy and Nanoscopy Facility has been partially supported by Fundació Cellex Barcelona. ; Peer reviewed

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. ; This work was supported by Grants PID2020-113371RB-C21 and PID2019-109390RB-I00 funded by MCIN/AEI/ 10.13039/501100011033 and by ERDF A way of making Europe, as well as by Grant CEX2018-000794-S funded by MCIN/AEI/ 10.13039/501100011033. Moreover, J.B. was supported by Grant PRE2018-083513 funded by MCIN/AEI/10.13039/501100011033 and by ESF Investing in your future. The work was also partially supported by the Catalan Government through the network of recognized research groups (2017 SGR_902 (DR, EP) and 2017SGR979 (RL-A, EE)). ; Peer reviewed

Acrylamide (ACR), a type-2 alkene, may lead to a synaptopathy characterized by ataxia, skeletal muscles weakness and numbness of the extremities in exposed human and laboratory animals. Currently, only the mildly affected patients undergo complete recovery, and identification of new molecules with therapeutic bioactivity against ACR acute neurotoxicity is urgently needed. Here, we have generated a zebrafish model for ACR neurotoxicity by exposing 5 days post-fertilization zebrafish larvae to 1 mM ACR for 3 days. Our results show that zebrafish mimics most of the pathophysiological processes described in humans and mammalian models. Motor function was altered, and specific effects were found on the presynaptic nerve terminals at the neuromuscular junction level, but not on the axonal tracts or myelin sheath integrity. Transcriptional markers of proteins involved in synaptic vesicle cycle were selectively altered, and the proteomic analysis showed that ACR-adducts were formed on cysteine residues of some synaptic proteins. Finally, analysis of neurotransmitters profile showed a significant effect on cholinergic and dopaminergic systems. These data support the suitability of the developed zebrafish model for screening of molecules with therapeutic value against this toxic neuropathy. © 2017 The Author(s). ; We thank Dr. Harold A. Burgess for providing Flote software and advice with the kinematic analysis. The 3A10 antibody, developed by Jessell, T.M./Dodd, J./Brenner-Morton, S. (DSHB Hybridoma Product 3A10), was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. This work was supported in part by the NATO SfP project MD.SFPP 984777 (D.R., A.A. and N.G.R.), the US Army Environmental Quality and Installations Research Program (N.G.R.), the Advanced Grant ERC-2012-AdG-320737 (D.R., C.G-C, R.T.), the Spanish Government (CTM2014-51985-R; D.R.) and the I-CORE Program of the Planning and Budgeting Committee and The Israel Science Foundation (grant No. 1775/12 to A.A.) for the purchase of the mass spectrometer. ; Peer reviewed

Catecholamine-triggered β-adrenoceptor (β-AR) signaling is essential for the correct functioning of the heart. Although both β1 - and β2 -AR subtypes are expressed in cardiomyocytes, drugs selectively targeting β1 -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 β1 -AR activation by means of photoswitchable drugs with a high level of β1 -/β2 -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 β1 -AR in native environments using light. ; We thank Ignacio Pérez (IQAC-CSIC, Barcelona), Yolanda Pérez (IQAC-CSIC, Barcelona), Lourdes Muñoz (SimChem, IQAC-CSIC, Barcelona) and Carme Serra (SimChem, IQAC-CSIC, Barcelona) for technical support. We thank Diana Baxter (Institute for Research in Biomedicine, IRB, Barcelona) for her thorough revision of the language of this manuscript. We thank Dr. Kees Jalink (The Netherlands Cancer Institute, Amsterdam, the Netherlands) for providing the plasmids encoding for the Epac-SH188 biosensor. We thank the University of Vic-Central University of Catalonia (UVic-UCC) and Dr. Marta Otero for the material assignment which helped in some biological assays. We thank Nikos Hatzakis for access to the Olympus IX81 confocal microscope (UCPH, DK). This work was supported by ERDF-FEDER European Fund and Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación (projects CTQ2017-89222-R and PID2020-120499RB-I00) and by the Catalan government (2017 SGR 1604) to A.L. X.R. research was financed by the ...

Organophosphorus compounds are acetylcholinesterase inhibitors used as pesticides and chemical warfare nerve agents. Acute organophosphorus poisoning (acute OPP) affects 3 million people, with 300 000 deaths annually worldwide. Severe acute OPP effects include overstimulation of cholinergic neurons, seizures, status epilepticus, and finally, brain damage. In a previous study, we developed 3 different chemical models of acute OPP in zebrafish larvae. To elucidate the complex pathophysiological pathways related to acute OPP, we used integrative omics (proteomic, transcriptomics, and metabolomics) on these 3 animal models. Our results show that these stochastic, apparently disparate morphological phenotypes can result from almost linear concentration-response variations in molecular levels. Results from the multiomics analysis strongly suggest that endoplasmic reticulum stress might play a central role in the pathophysiology of severe acute OPP, emphasizing the urgent need of further research on this molecular pathway. Endoplasmic reticulum stress could be an important therapeutic target to be included in the treatment of patients with severe acute OPP. ; NATO SfP project MD.SFPP 984777 (D.R.); the European Research Council under European Union's Seven Framework Programme (FP/2007–2013)/ERC Grant Agreement No. 320737; the Spanish Government (CTM2017-83242-R and CTM2015-65691-R). M.F. acknowledges the financial support from the Government of Catalonia through a Beatriu de Pinos fellowship (2016 BP-B 00233) ; Peer reviewed

Inhibition and aging of neuropathy target esterase (NTE) by exposure to neuropathic organophosphorus compounds (OPs) can result in OP-induced delayed neuropathy (OPIDN). In the present study we aimed to build a model of OPIDN in adult zebrafish. First, inhibition and aging of zebrafish NTE activity were characterized in the brain by using the prototypic neuropathic compounds cresyl saligenin phosphate (CBDP) and diisopropylphosphorofluoridate (DFP). Our results show that, as in other animal models, zebrafish NTE is inhibited and aged by both neuropathic OPs. Then, a neuropathic concentration inhibiting NTE activity by at least 70% for at least 24 h was selected for each compound to analyze changes in phosphatidylcholines (PCs), lysophosphatidylcholines (LPCs) and glycerolphosphocholine (GPC) profiles. In spite to the strong inhibition of the NTE activity found for both compounds, only a mild increase in the LPCs level was found after 48 h of the exposure to DFP, and no effect were observed by CBDP. Moreover, histopathological evaluation and motor function outcome analyses failed to find any neurological abnormalities in the exposed fish. Thus, our results strongly suggest that zebrafish is not a suitable species for the development of an experimental model of human OPIDN. © 2018 The Author(s). ; This study was funded by the NATO SfP project MD.SFPP 984777 (D.R.), the European Research Council under European Union's Seven Framework Programme (FP/2007–2013)/ERC Grant Agreement n. 320737, and the Spanish Government (CTM2017-83242-R; D.R.). M.F. acknowledges the financial support from the Government of Catalonia through a Beatriu de Pinos fellowship (2016 BP-B 00233). I.F. was supported by the Ministry of Economy and Competitivity of Spain (FPI grant BES-2015-075023). The authors thank Mr. Marc Mañas for his valuable assistance building the Open Field Test setup. ; Peer reviewed