Abstract

Classical cases of developmental neurotoxicity (DNT) in humans and advances in risk assessment methods did not prevent the emergence of new chemicals with (suspected) DNT potential. Exposure to these chemicals may be related to the increased worldwide incidence of learning and neurodevelopmental disorders in children. DNT is often investigated in a traditional manner (in vivo using large numbers of experimental animals), whereas development of in vitro methods for DNT reduces animal use and increases insight into cellular and molecular mechanisms of DNT. Several essential neurodevelopmental processes, including proliferation, migration, differentiation, formation of axons and dendrites, synaptogenesis, and apoptosis, are already being evaluated in vitro using biochemical and morphological endpoints. Yet, investigation of chemical-induced effects on the development of functional neuronal networks, including network formation, inter- and intracellular signaling and neuronal network function, is underrepresented in DNT testing. This view therefore focuses on in vitro models and innovative experimental approaches for functional DNT testing, ranging from optical and electrophysiological measurements of intra- and intercellular signaling in neural stem/progenitor cells to measurements of network activity in neuronal networks using multielectrode arrays. The development of functional DNT assays will strongly support the decision-making process for measures to prevent potential chemical-induced adverse effects on neurodevelopment and cognition in humans. We therefore argue that for risk assessment, biochemical and morphological approaches should be complemented with investigations of neuronal (network) functionality.

The developing brain is a sensitive target for chemical disruption (Rice and Barone, 2000). Epidemiological studies have demonstrated that exposure of the developing nervous system to neurotoxicants, such as lead and polychlorinated biphenyls, results in adverse effects in humans. These include developmental delays or alterations in behavior, cognition, and motor functions (Winneke, 2011). However, these classical cases of developmental neurotoxicity (DNT) in humans and advances in risk assessment methods did not prevent the emergence of new (suspected) developmentally neurotoxic chemicals. Over the past three decades, the incidence of learning and neurodevelopmental disorders in children appears to be increased (Herbert, 2010). Exposure to environmental chemicals has been identified as a risk factor for these neurodevelopmental disorders (Bellinger, 2012). Despite these findings, DNT testing is not a primary objective in chemical testing programs.

DNT is often investigated in a traditional manner: in vivo using large numbers of experimental animals, mostly rodents (OECD, 2007). The development of in vitro methods for DNT can play a role in the reduction of animal use while increasing time- and cost-effectiveness. Moreover, these methods have the potential to provide insight into cellular and molecular mechanisms of DNT that are not readily identified in experimental animal studies. Many efforts are currently undertaken in the field of (developmental) neurotoxicity to improve the predictivity of in vitro models by increasing their sensitivity and specificity (e.g., Lein et al., 2007).

During development, the brain evolves from embryonic ectodermal cells to a complex network of many specialized, highly interconnected and structured cells. Development continues after birth and developmental processes need to take place within a strictly controlled time frame and in the right order. The DNT of a chemical is therefore not only related to the dose and whether or not the chemical (or its metabolites) can reach the nervous system, but also to the developmental phase during which the brain is exposed (Rice and Barone, 2000). The specific vulnerability of the developing brain is also related to the lower capacity of the blood-brain barrier to protect the brain from xenobiotics (Ek et al., 2012). Although the regenerative capacity of the developing brain may allow for adaptation to or compensation for chemical-induced effects, exposure during key periods of brain development may also result in enhanced susceptibility at a later age (e.g., Eriksson and Talts, 2000).

The rapid development and marketing of new chemicals constantly alters the human exposure situation. In several cases, exposure reduction measures have been necessary for a particular chemical after identification of adverse effects in children. To avoid such situations in the future, the development of screening methods for potential developmentally neurotoxic chemicals is critical. In this view, we therefore support the implementation of in vitro assays to identify and prioritize cellular and molecular mechanisms of DNT, and we advocate the inclusion of functional parameters. Currently, no guidelines on in vitro DNT models and tests exist, but several in vitro testing approaches have been initiated (Bal-Price et al., 2010; Coecke et al., 2007; van Thriel et al., 2012).

For the investigation of chemical-induced effects on the (developing) brain, specific cellular processes can be investigated using in vitro models. These cellular processes are critical for the formation of functional neuronal networks that receive, conduct, and transmit signals via chemical or electrical synapses, and relay information between specific brain regions for information processing as well as learning and memory formation. Essential neurodevelopmental processes include proliferation, migration, differentiation, formation of axons and dendrites, synaptogenesis, myelination, and apoptosis (Lein et al., 2007; Rice and Barone, 2000). Many of these parameters are already being evaluated in vitro using biochemical and morphological endpoints (Table 1[A]). However, investigation of chemical-induced effects on the development of neuronal network functionality is underrepresented in DNT testing (< 20% of original research articles in a PubMed search for in vitro DNT are related to neuronal function; for details see Supplemental Material). Parameters to investigate in neuronal network functionality include network formation, action potential generation, calcium homeostasis, synaptic transmission, and synaptic plasticity (Table 1[B]). In this view, approaches and recent innovations are reviewed with emphasis on those that allow for investigation of chemical-induced effects on the development of functional neuronal networks that form the basis for (human) cognition.

TABLE 1

In Vitro DNT Testing

 Process In vitro endpoint Example approach 
(A) Structure Proliferation Cell viability Biochemistry 
  Apoptosis Biochemistry 
 Migration Migration distance (High content) image analysis 
  Cytoskeletal proteins Fluorescent imaging 
   Gene/protein expression 
 Differentiation Neurotransmitter receptors Immunocytochemistry 
   Gene/protein expression 
  Cell-specific markers Immunocytochemistry 
  Enzyme activity Biochemistry 
   Gene/protein expression 
  Neurite outgrowth (High-content) image analysis 
  Myelination Gene/protein expression 
 Synaptogenesis Synaptic proteins Immunocytochemistry 
  Synaptic connections (High-content) image analysis 
(B) Function Intracellular signaling Calcium concentration Fluorescent imaging 
  Oxidative stress Fluorescent imaging 
  Kinase signaling Biochemistry 
  Ion channel function Electrophysiology 
  Membrane potential Fluorescent imaging 
   Electrophysiology 
 Intercellular signaling Neurotransmitter release Electrophysiology 
   Fluorescent imaging 
  Neurotransmitter receptors Electrophysiology 
 Network function Network activity Electrophysiology 
  Synaptic plasticity Electrophysiology 
 Process In vitro endpoint Example approach 
(A) Structure Proliferation Cell viability Biochemistry 
  Apoptosis Biochemistry 
 Migration Migration distance (High content) image analysis 
  Cytoskeletal proteins Fluorescent imaging 
   Gene/protein expression 
 Differentiation Neurotransmitter receptors Immunocytochemistry 
   Gene/protein expression 
  Cell-specific markers Immunocytochemistry 
  Enzyme activity Biochemistry 
   Gene/protein expression 
  Neurite outgrowth (High-content) image analysis 
  Myelination Gene/protein expression 
 Synaptogenesis Synaptic proteins Immunocytochemistry 
  Synaptic connections (High-content) image analysis 
(B) Function Intracellular signaling Calcium concentration Fluorescent imaging 
  Oxidative stress Fluorescent imaging 
  Kinase signaling Biochemistry 
  Ion channel function Electrophysiology 
  Membrane potential Fluorescent imaging 
   Electrophysiology 
 Intercellular signaling Neurotransmitter release Electrophysiology 
   Fluorescent imaging 
  Neurotransmitter receptors Electrophysiology 
 Network function Network activity Electrophysiology 
  Synaptic plasticity Electrophysiology 

Notes. Key neurodevelopmental processes in the structural (A) and functional (B) development of functional neuronal networks, corresponding in vitro endpoints and example approaches for DNT studies. For a table including references, readers are referred to the Supplementary data.

IN VITRO MODELS FOR DNT TESTING

Various in vitro systems, including cell lines, primary cell cultures, and stem cell models, are used to model the developing nervous system. Immortalized neuronotypic cell lines provide a homogeneous cell population that is often well characterized and controlled. In many cases, they are easily cultured, divide rapidly, and can be derived from different species, including humans. Compared with primary cells, cell lines readily allow the incorporation of exogenous genes. Additionally, some of these cells can be differentiated into different neural subtypes (Costa et al., 2011). However, their genetic stability decreases with increasing number of passages, neurites do not always represent true axons or dendrites and functional synapses are often absent (Breier et al., 2010). Additionally, transformed cell lines may represent only a subset of cells and may not have the same (functional) phenotype as primary cells.

Primary cells usually have the same phenotype as brain cells in vivo and maintain most neurodevelopmental processes. They can be isolated relatively easily from distinct brain areas and at different developmental stages (Breier et al., 2010; Costa et al., 2011). However, these cultures often contain populations of postmitotic neurons, which is a disadvantage for the study of neurodevelopmental processes. Practical limitations include a relatively short life span, variability between cultures, and the limited availability of certain tissues (e.g., from human fetuses and/or surgical patients; Breier et al., 2010; Costa et al., 2011).

Stem cells, which are characterized by their ability to self-renew by proliferation, are abundantly present in the developing nervous system and give rise to the different cell types in the brain. Therefore, (cord blood-derived) embryonic stem cells (ESCs) and neural progenitor cells (NPCs) are particularly suited for the investigation of neurodevelopmental processes (Breier et al., 2010; Buzanska et al., 2009; Davila et al., 2004). ESCs are pluripotent, and they can give rise to (almost) all cell types of the organism, whereas multipotent progenitor cells already have some characteristics from the region from which they are isolated. As a result, (neuronal) progenitor cells give rise to tissue-specific cell types (Breier et al., 2010; Davila et al., 2004). Therefore, NPCs may be more efficiently applied to generate a heterogeneous neural culture than pluripotent ESCs, because ESCs first need to differentiate into a neural phenotype. Also, NPC cell lines with differentiation potential have been generated (e.g., Donato et al., 2007). It is not yet firmly established whether ESC/NPCs can form functional neuronal networks, though recent findings indicate that ESCs develop functional neuronal characteristics in vitro (Heikkilä et al., 2009; Weick et al., 2011, Zimmer et al., 2011). In most cases, however, ESCs require several weeks of differentiation before functional neuronal (network) characteristics develop, which is a limiting factor for the throughput capacity in a toxicological setting.

Neuronal network function depends critically on the presence of multiple neural cell types (Fellin, 2009), including neurons, oligodendrocytes, microglia, and astrocytes. The importance of the presence of multiple neural cell types for DNT testing depends on the parameter of interest. For example, acute effects on neurotransmitter release can be investigated in a homogenous cell culture, but effects on cell viability will vary between different cell types and heterogeneous cell models, for example due to variations in their antioxidant capacity (Giordano et al., 2009) and receptor profile. As mentioned earlier, the DNT potential of a chemical can vary depending on the stage of brain development during which an organism is exposed. This specific characteristic is not fully represented in most in vitro models, but can potentially be improved in heterogeneous models and using subchronic (developmental) exposure scenarios.

EXPERIMENTAL APPROACHES FOR DNT TESTING

Biochemical and Morphological DNT Testing

In in vitro DNT studies, basal neurodevelopmental processes such as proliferation, migration, and differentiation are commonly assessed (Table 1[A]). These parameters are being investigated in different cell models, including NPCs. These cells can be cultured in monolayers or as free-floating spheres and proliferate in the presence of the appropriate growth factors. In spheres, withdrawal of growth factors triggers cells to migrate out of the sphere onto an extracellular matrix and differentiate into different cell types expressing neuronal and glial markers (Breier et al., 2010; Moors et al., 2009). As such, effects of chemicals on proliferation and migration distance can be measured. Additionally, effects on the presence of different cell types in the migrated network can be determined, e.g., by investigating marker proteins such as nestin, βIII-tubulin, and glial fibrillary acidic protein for developing and mature neurons and astrocytes, respectively (Kuegler et al., 2010).

Most in vitro DNT studies describe effects of chemicals on cell viability in neural cell types. When based on cell viability only, the distinction between general toxicity and (developmental) neurotoxicity remains unclear. Nevertheless, dose range finding for the investigation of specific DNT parameters should, in all cases, be included to prevent confounding by effects on cell viability. Mitochondrial function (including the generation of ATP, metabolism of reactive oxygen species, and modulation of calcium homeostasis) is not only essential for neuronal viability and function (including plasticity), but also plays an important role in the structural development (e.g., by regulating apoptosis) of neuronal networks (Cheng et al., 2010). Endpoints of mitochondrial function in isolated mitochondria or intact (neural) cells that may be used in in vitro DNT include mitochondrial respiration, ATP turnover, and proton leak (reviewed in Brand and Nicholls, 2011).

Structural aspects of functional network development include neurite outgrowth (i.e., the development of axons and dendrites) and synaptogenesis. Several studies have demonstrated the ability of in vitro models to detect chemically induced changes in neurite outgrowth, using manual or (semi)automated methods to acquire microscopic images and quantify neurite development (Radio and Mundy, 2008; Radio et al., 2010).

Recently, systems have been developed for simultaneous investigation of multiple endpoints. An example is the imaging-based quantification of neurite growth in LUHMES human neuronal precursor cells. This approach allows for the simultaneous evaluation of cell viability and neurite outgrowth, thereby identifying specific neuritotoxic chemicals (Stiegler et al., 2011). Moreover, high content image analysis (HCA) platforms have been developed to track phenotypic changes in individual cells using different fluorescent labels in a multiwell format. As such, HCA allows for the investigation of chemical-induced effects on multiple morphological endpoints in neuronal (network) development over time, including neuron density, neurite length, and the number of synapses (Harrill et al., 2011b; Radio et al., 2010).

Neuronal (Network) Function in DNT Testing

In functional neuronal networks, intercellular communication takes place at the synaptic connections, whereas intracellular signaling cascades, such as calcium signaling, modulate neuronal signals. Besides affecting a multitude of (sub)cellular processes, calcium is the trigger of vesicular neurotransmitter release in neuronal cells through activation of the exocytotic release machinery (Neher and Sakaba, 2008). Chemical-induced changes in intracellular calcium dynamics, e.g., by effects on the influx, efflux, or compartmentalization of calcium, can alter neurotransmitter release, thereby modulating intercellular communication. Changes in intracellular calcium levels can be investigated using fluorescent markers, preferably at high spatial (single-cell) and temporal resolution (Heusinkveld and Westerink, 2011).

Changes in neurotransmitter release can be investigated using amperometry or other electrophysiological techniques (Westerink, 2004) as well as optical techniques, e.g., imaging of FM dyes (Brumback et al., 2004) or fluorescent false neurotransmitter (Gubernator et al., 2009). For intercellular communication, proper function of (mainly postsynaptic) neurotransmitter receptors is also critical. Several chemicals have been shown to modulate neurotransmitter receptor function (Atchison, 1988), potentially resulting in altered neuronal network function. Receptor function and chemical-induced effects thereon can be studied in vitro by electrophysiological measurements. To this aim, high-throughput patch clamp systems (e.g., Spencer et al., 2012), which are at this moment mostly used in a drug development setting, could be applied in (developmental) neurotoxicity testing.

The (development of) intercellular communication in in vitro neuronal networks can be investigated using fluorescent dyes for monitoring membrane potential (Wolff et al., 2003) or more directly using functional electrophysiological recordings, e.g., using multielectrode arrays (MEAs). MEA systems typically consist of a cell culture surface with an integrated array of microelectrodes, allowing simultaneous extracellular recording of electrical activity at different individual sites in a neuronal network. This can be used to study (the development of) spontaneous activity patterns as well as evoked activity (Johnstone et al., 2010). Importantly, the latter also allows for the investigation of synaptic plasticity (activity dependent synaptic efficiency), which is critical for learning and memory formation (Citri and Malenka, 2008). In vitro synaptic plasticity is generally investigated in ex vivo brain slices, in particular hippocampal slices using standard protocols (Hernandez et al., 2005). Investigation of plasticity in neural cells cultured on MEAs (e.g., Chiappalone et al., 2008) could improve throughput for chemical testing, although a standard protocol for the investigation of (chemical-induced effects on) plasticity in a neurodevelopmental context remains to be developed.

Chemical-induced changes in network function measured in a MEA system may be due to changes in electrical activity as well as in the release or receival of intercellular signals. MEA systems thus provide an integrated, but not pathway specific, measure for effects on neurotransmission that can be applied for DNT screening purposes. MEA approaches have only relatively recently been introduced in neurotoxicological research, mostly for the investigation of acute disruption of network function. Recent investigations of the interlaboratory variation in MEA recordings in rat cortical cultures have shown that neurotoxic responses can be measured reproducibly (Novellino et al., 2011). Only very recently, MEA research has been initiated for the investigation of DNT, demonstrating the development of function in neuronal cultures (Hogberg et al., 2011; Robinette et al., 2011). To improve the throughput of MEA systems, a multiwell MEA was recently introduced and tested with a training set of chemicals for its application in neurotoxicity testing (McConnell et al., forthcoming). This higher throughput format may further increase the potential of MEA systems in (developmental) neurotoxicity testing.

DISCUSSION

Recent approaches and innovations for the in vitro investigation of chemical-induced effects on neurodevelopmental processes allow for investigation of functional endpoints (Table 1[B]). These endpoints, such as calcium homeostasis, neurotransmitter release, and intercellular communication in neuronal networks, reflect the actual function of the nervous system and are therefore a valuable addition to biochemical and morphological endpoints. Other important aspects in the development of the nervous system, e.g., myelinization, are largely unexplored in (developmental) neurotoxicity studies. It is also notable that in vitro neuronal cultures have low metabolic capacity. Specific impact of metabolites therefore has to be investigated separately, e.g., by directly exposing the cells to the metabolites of interest.

The maintenance of newly formed synapses depends on their activity. Functional neuronal processes that play a role in interneuronal communication (e.g., calcium signaling, neurotransmitter release, and neurotransmitter receptor function) are therefore not only the ultimate goal of functional neuronal network formation, but are also critically involved in the developmental process itself. As such, acute neurotoxicity may also affect brain development, depending on the timing of exposure to a neurotoxic chemical. The predictivity of acute neurotoxicity for DNT potential is however not yet characterized.

Noteworthy, recommendations for the development of screening and prioritization methods for DNT as well as a draft list of model chemicals were recently provided (Crofton et al., 2011). It should also be noted that most of the approaches highlighted in this view originate from the neuroscience field. Therefore, emerging techniques and innovations in neuroscience should be closely followed and assessed for their potential and applicability in (developmental) neurotoxicity studies.

For a large number of high-production chemicals, basic and more specific toxicological data are currently lacking (Lein et al., 2007). Replacing animal experiments with in vitro models already allows for higher testing capacity. In view of the large number of chemicals to be tested, the use of medium-to-high throughput approaches is, in general, preferable for chemical screening purposes for (functional) DNT effects. Most biochemical and structural assays for in vitro DNT testing have the potential for medium-to-high throughput testing and there are cases where medium-to-high throughput testing was able to confirm the (developmental) neurotoxic potential of well-known DNT chemicals (e.g., Breier et al., 2008; Culbreth et al., forthcoming; Harrill et al., 2011a; Radio et al., 2008). However, achieving this level of throughput is often challenging for functional analysis and in-depth investigation of DNT mechanisms. Additionally, the generation of large amounts of data, either by testing many chemicals using high-throughput approaches or generating high density data (e.g., using MEAs, HCA, or optical recordings) brings forth new requirements in data storage and analysis for which the implementation of bioinformatics is necessary.

In vitro assays are useful to regulators involved in hazard and risk assessment and legislation of chemicals, as well as to chemical and pharmaceutical industries that may want to apply in-house DNT screening assays in chemical and drug development. However, due to the complexity of the developing (central) nervous system and considerations such as exposure timing, in vitro models are not likely to completely replace in vivo DNT testing. Nevertheless, there is general agreement within the field of toxicology that the future of toxicity testing is a tiered testing strategy that focuses on pathways that are critical for adequate functioning of cells, organs, and organisms (Bal-Price et al., 2010, Crofton et al., 2011; Seiler et al., 2011). This includes the integration of different testing strategies for exposure and toxicity with emphasis on nonanimal models, including integrated genomic and proteomic analyses of chemical-induced effects on signaling pathways (e.g., Pennings et al., 2012), developmental processes, and the formation of functional neuronal networks (e.g., Slotkin and Seidler, 2010).

Apart from being adverse on an individual level, neurodevelopmental disorders have economic consequences due to reduced potential to contribute to society as well as additional costs for medical care. Low-dose exposure to chemicals has the potential to cause small but population-wide adverse effects on cognition and incidence rates of neurodevelopmental disorders in humans. The development of screening assays for DNT is critical for the decision-making process to prevent this. We therefore argue that for risk assessment of chemical-induced effects on human neurodevelopment and cognitive function, biochemical and morphological approaches should be complemented with investigations of neuronal network functionality.

SUPPLEMENTARY DATA

Supplementary data are available online at http://toxsci.oxfordjournals.org/.

FUNDING

European Union (DENAMIC; FP7-ENV-2011-282957); The Netherlands Organization for Health Research and Development (ZonMW; 85300003); Faculty of Veterinary Medicine of Utrecht University.

ACKNOWLEDGMENTS

We apologize to all authors of primary literature or previous reviews on (functional) in vitro models for DNT that we could not include due to space limitations. The authors declare they have no actual or potential competing financial interests.

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