The Neurotoxicology Research Group investigates the cellular and molecular mechanisms of action of toxic substances (food-related products, health products, drugs and environmental contaminants) on the nervous system.
Research focuses on adverse effects of toxic substances on neuronal network activity, membrane signalling through membrane receptors and ion channels, and on cellular communication through exocytosis. Additionally, intracellular signalling cascades and neurodevelopment are studied for further hazard characterization of various classes of environmental toxicants.
Our research projects focus on those exposures that are associated with strong societal concern (e.g., alternative flame retardants, drugs of abuse and electromagnetic fields) and/or neurodegenerative disorders, such as Parkinson's Disease (e.g., pesticides and chemical mixtures) and/or developmental neurological disorders, such as ADHD/ASD (chemical mixtures of pesticides and persistent organic pollutants).
Within all research projects our focus is on the use of in vitro systems and alternatives to animal use, including models for cell-cell communication (neurotransmission) and for studying developmental aspects of toxicity.
Primary cortical cultures are the current standard to capture the complexity of the nervous system in an in vitro model for neurotoxicity testing. These cortical cultures are heterogeneous and consist of different types of inhibitory GABA-ergic and excitatory glutamatergic neurons and supportive cells, e.g. astrocytes. In vitro, these cells grow into complex network of specialized, highly connected and structured cells that develop spontaneous network activity after 1 week in culture (Dingemans et al., 2016). As such, these cortical cultures are useful for investigating effects of chemicals on (the development of) neuronal (network) functionality, changes in intracellular calcium levels and/or neurotransmitter release (de Groot et al., 2016; Duarte et al., 2017; Hondebrink et al., 2016, 2017). Recently, cortical cultures grown on multi-electrode arrays have been used for food safety testing (Kasteel and Westerink, 2017; Nicolas et al., 2014).
The latest development is the use of human induced pluripotent stem cells (hiPSCs) as model for neurotoxicity testing. One particular advantage of the use of hiPSCs is the elimination of the need for interspecies translation. Multiple types of human hiPSCs are available that can be used to assess specific types of toxicity (for example by using dopaminergic neurons) and/or grow into sophisticated networks of mixed neuronal nature. These hiPSC-derived, spontaneously active, neuronal networks effectively capture the complexity of the nervous system and represent the current state of the art for in vitro neurotoxicity testing. Currently, human iPSCs are used in several of our projects to investigate the effects of chemicals/drugs on (the development of) neuronal (network) functionality, changes in intracellular calcium levels and/or neurotransmitter release (Hondebrink et al., 2017; Kasteel and Westerink, 2017; Tukker et al., 2016; Tukker er al., 2018).
In our research projects, a range of techniques is used to assess the neurotoxic potency of test compounds. Multi-well Micro-Electrode Arrays (mwMEAs) consist of a cell culture surface with an integrated microelectrodes array that allows for simultaneous recordings of spontaneous electrical activity at several locations in neuronal networks in vitro, thereby providing an integrated measure for chemical-induced effects on (the development of) neurotransmission within a neuronal network in vitro. We use mwMEA recordings for (sub)acute and chronic neurotoxicity, developmental neurotoxicity and neurodegeneration studies (see e.g. Dingemans et al., 2016; de Groot et al., 2013, 2014). Recently, we used this approach for food safety testing (Kasteel and Westerink, 2017; Nicolas et al., 2014) and hazard characterization of (illicit) drugs (Hondebrink et al., 2016, 2017). Moreover, it can also be applied for cardiotoxicity testing (Zwartsen et al., 2019).
To exclude that neurotoxic effects are simply due to cytotoxicity, we routinely use a MTT assay (e.g. Dingemans et al., 2009; Hondebrink et al., 2009) or a combined alamar Blue (aB) and Neutral Red (NR) assay(e.g. Hendriks et al., 2012; Heusinkveld et al., 2010; Dingemans et al., 2010) to determine whether cell viability is affected by chemical exposure.
Additional techniques are available to investigate effects of test compounds on calcium homeostasis, which is essential for neuronal function. We therefore routinely measure changes in the intracellular calcium concentration using single cell fluorescent microscopy (see e.g., de Groot et al., 2014; Dingemans et al., 2010; Heusinkveld et al., 2010, 2016, 2017; Hondebrink et al., 2011; Meijer et al., 2014, 2015).
Spontaneous activity in rat neonatal cortical cultures (left) or human iPSC-derived neuronal cultures (middle) can be measured in 48-wells MEA plates from 4-21 days in vitro using a MEA platform (right). Neuronal activity can be visualized as ‘activity maps’ per well as well as mean spike rate per electrode, resulting in concentration-effect curves that demonstrate the effect of neuroactive compounds (including (environmental) chemicals, drugs, food components) on neuronal activity.