Elucidating mechanisms of eco-neurotoxicity
The eco-neurotoxic properties are often analysed using quantitative behavioural phenotyping. Perturbations in behavioural phenotypes are increasingly being linked to diminished ecological fitness at environmentally relevant concentrations of pollutants, in both acute and chronic exposure scenarios.
Despite the mounting interest in neuro-behavioural phenotyping as a toolbox for regulatory chemical risk assessment, there is a considerable paucity in deciphering of molecular foundations of eco-neurotoxicity. The available behavioural tests enable detection of subtle alterations in animal responses induced by low levels of toxic factors. They, however, do not provide sufficient data on the cellular and molecular mechanisms of such changes.
Due to relative simplicity of their central nervous systems, many small aquatic organisms are particularly well suited to explore molecular mechanisms of eco-neurotoxicity.
Currently, there is a growing number of molecular methods that can be applied to studying the mechanistic basis of behavioural changes in aquatic animals
To understand the mechanisms of eco-neurotoxicity we employ a battery of basic as well as advanced neuro-behavioural phenotyping combined with diverse molecular and physiological methods as well as transgenic zebrafish models.
Programmed cell death (PCD)
The neurotoxicants can damage the discrete neuronal sub-populations as a result of specific or non-specific (e.g. generation of reactive oxygen species, damage to glial cells, etc) effects. Pollutant-induced neuronal cell death during neuro-development often trigger functional deficiencies in behavioural phenotypes.
Particular interest in biology of cell death comes from the discovery of multitude complex regulatory circuits that control the cellular demise. Historically cells were known die in two distinct morphologically and biochemically processes such as apoptosis and necrosis often referred to as programmed and accidental cell death, respectively.
However other alternative quasi-apoptotic (e.g. caspase-independent apoptosis, necroptosis) and nonapoptotic (e.g. autophagy) mechanism discovered lately may also be involved in pollutants mode of action.
We employ diverse methods that allow for implementation of apoptotic assays on live suspension, adherent cells, in situ using transgenic models and innovative fluorescent probes as well as fixed specimens such as cell and tissue samples.
Markers for many diverse modes of caspase-dependent cell death have been particularly well developed in aquatic vertebrate models and can be analysed using techniques ranging from molecular techniques, microscopy and flow cytometry as well as imaging cytometry.
Diverse modes of programmed cell death can be initiated in many organs by exposure to pollutants. This can lead to many developmental as well functional patho-physiologies. An example of mitochondrial pathway of caspase-dependent apoptosis commonly triggered by chemical damage is shown above.
Analysis of cytotoxicity and programmed cell death can be rapidly performed using flow cytometry as well as imaging cytometry.
(Left) A flow cytometer BD FACSCalibur (Right) Discrimination of live, apoptotic, and late apoptotic/necrotic cells using Annexin V‐FITC/PI and FLICA/PI bioassays. Green events (R1): live cells; blue events (R2): apoptotic cells; red events (R3): late apoptotic/necrotic cells (PI‐positive). Cell debris showing extremely low FSC/SSC values were excluded electronically.
Analysis of discrete neuronal sub-populations
We employ in situ hybridisation and whole mount immuno-histochemistry labelling with fluorescently labelled antibodies against markers of neuronal sub-populations. Such analysis is usually performed using quantitative fluorescence microscopy and/or imaging cytometry and enables characterisation of specific neuronal sub-populations as well as morphologies.
Gross majority of neuronal markers have been recently developed, characterized and successfully validated in zebrafish models. A significant repertoire of neuronal markers has been also characterised in freshwater planarian models and conservation of synaptic transmission enables visualisation of discrete sub-populations in many invertebrates.
Recent advances in transgenic zebrafish models provide also innovative and non-invasive approaches to rapidly perform time-resolved fluorescent imaging in living specimens under mild anaesthesia.
Analysis of discrete neuronal sub-populations.
A significant repertoire of neuronal markers has been characterised in zebrafish and freshwater planarian models enabling visualisation of discrete sub-populations.
Alterations in gene expression of neuronal-specific genes, can indicate sensitive alterations in the central nervous system that can be correlated with alterations of behavioural responses at the molecular level.
Analysis of transcript expression is routine at a small scale using quantitative, reverse polymerase chain reaction (QRT-PCR) as well as global expression profiling with gene microarray and next-generation sequencing technologies.
Such analysis can determine changes in expression patterns in neuronal stem cells and/or in developing neurons.
Analysis of transcript expression is routine at a small scale using quantitative, reverse polymerase chain reaction (QRT-PCR) as well as global expression profiling with gene microarray and next-generation sequencing technologies
Functional analysis of neuronal transmissions
Functional analysis of neuronal transmission is one of the fundamental approaches to explain the mechanisms of neuro-behavioural changes in organisms exposed to neurotoxicants.
Historically the standard marker of neurotoxicity has been activity of acetylcholinesterase (AChE), a key enzyme in the degradation of acetylcholine in neuromuscular synapses. Activity of AChE, measured using the Ellman’s spectrophotometric method, can be used to test if locomotor alterations upon toxicant exposures are associated by altered neuromuscular neurotransmission. This assay is particularly relevant to toxicants directly impacting activity of AChE such as such as organophosphate and carbamate pesticides.
ELISA (Enzyme-Linked Immunosorbent Assay) assays provide another useful toolbox for rapid and quantitative analysis of key signalling elements involved in neuronal transmission such as: receptors, neurotransmitters, mediators and enzymes regulating functions of the nervous system.
Recently the advances in using Mass Spectroscopy-High Performance Liquid Chromatography (Tandem MS-HPLC) have enabled rapid quantitative analysis of panels of neurotransmitters, metabolites and neuro-precursors opening new vistas for ecotoxicology and mechanistic neurotoxicology.
Our laboratory is currently also exploring applications of novel, non-invasive electro-physiological methods for real-time measuring neural activity in living small model organisms using multi-electrode array (MEA) technologies.
MEAs were developed for assessment of neuronal excitability using neural networks created from human induced pluripotent stem cells (iPSC). However there can be also used for non-invasive and label-free whole organism electrophysiology recordings on small organisms such as for instance living zebrafish larvae. Such innovative technologies enable powerful functional bioassays for supplementing neuro-behavioural tests in aquatic ecotoxicology, neurotoxicology and neuropharmacology.
Functional analysis of synaptic transmission
Activity of acetylcholinesterase (AChE), a key enzyme in the degradation of acetylcholine in neuromuscular synapses, measured using the Ellman’s spectrophotometric method has been a standard marker of neurotoxicity. Recently advances in using Mass Spectroscopy-High Performance Liquid Chromatography (Tandem MS-HPLC) enable rapid quantitative analysis of panels of neurotransmitters, metabolites and neuro-precursors opening new vistas for ecotoxicology and mechanistic eco-neurotoxicology. (Source: Tufi et al Environ. Sci. Technol. 2016, 50, 3222−3230)
Non-invasive in situ assessment of neuronal excitability in living small model organisms using microelectrode arrays (MEA)
Applications of novel, non-invasive electro-physiological methods for real-time measuring neural activity in living small model organisms using multi-electrode array (MEA) technologies enable powerful functional bioassays for supplementing neuro-behavioural tests in aquatic ecotoxicology, neurotoxicology and neuropharmacology. (Source: Axion BioSystems)