GABA type-A receptors (GABA_AR) and strychnine-sensitive glycine receptors (GlyR) are ligand-gated chloride ion channels that mediate inhibitory neurotransmission in the central nervous system (CNS). In adult neurons, GABA_AR and GlyR ion channels conduct an inhibitory anion current, mainly carried by chloride (Cl⁻) upon activation by γ-aminobutyric acid (GABA) and the amino acid glycine, respectively. In embryonic neurons however, due to a higher intracellular chloride concentration compared to adult neurons, receptor activation causes an outward directed, depolarizing and excitatory Cl⁻ flux (Webb and Lynch, 2007). GABA_ARs and GlyRs are both members of the pentameric ligand-gated ion channel (pLGIC) family and require five subunits to form a single functional oligomer. For GABA_ARs there are 19 genes known (α1–6, β1–3, γ1–3, δ, ε, θ, π, and ρ1–3) exhibiting a broad range of heterogeneity and many hundreds of theoretically possible subunit combinations (Olsen and Sieghart, 2009). For GlyRs there are four genes known (α1–3, β) in humans, exhibiting far less diversity compared to GABA_ARs (Lynch, 2009). Each GABA_AR or GlyR isoform as well as each subunit combination has a unique physiological and pharmacological profile. The subunit combination can change during development, in a tissue-specific manner or as a consequence of pathophysiological events (Lynch, 2004; Webb and Lynch, 2007; Esmaeili and Zaker, 2011; Rudolph and Möhler, 2014; Deidda et al., 2015). Genetic or molecular perturbation of the channels' function has been associated with severe neurological disorders including neuropathic pain (Lian et al., 2012; Xiong et al., 2012; Chen et al., 2014), chronic pain sensitization (Harvey et al., 2004; Zeilhofer, 2005; Lynch and Callister, 2006), hyperekplexia (Chung et al., 2010; Bode and Lynch, 2014), epilepsy (Meier et al., 2005; Eichler et al., 2008, 2009; Macdonald et al., 2010), fragile X mental retardation syndrome (D'Hulst et al., 2006), learning and memory deficits (Deidda et al., 2015) as well as neurodegeneration (Kang et al., 2015). In addition, GABA_ARs and GlyRs are increasingly acknowledged in the context of immunomodulation (Stoffels et al., 2011; Gunn et al., 2015), amyotrophic lateral sclerosis (Martin and Chang, 2012) and cancer (Neumann et al., 2004; Cuddapah and Sontheimer, 2011). Therefore, GABA_ARs and GlyRs, including individual isoforms as well as the various subunit combinations in their native neuronal environment are increasingly considered highly attractive drug targets for therapeutic intervention (Alexander et al., 2015). Due to their fundamental role in inhibitory neurotransmission GABA_ARs and GlyRs are increasingly recognized in the context of neurotoxicity (Suñol et al., 1989; Hall and Hall, 1999; Narahashi, 2002; Vale et al., 2003; Mohamed et al., 2004; Islam and Lynch, 2012) and in vitro neurotoxicity testing (NT) (Talwar et al., 2013; Tukker et al., 2016).

Although GABA_ARs and GlyRs play fundamental roles during brain development (Avila et al., 2013, 2014), these receptors have only sparsely been associated with developmental neurotoxicity and developmental neurotoxicity (DNT) testing. This is even more surprising as the incidence of neurological diseases including learning and developmental disorders has increased in recent years (May, 2000; Colborn, 2004; Rauh et al., 2006; Herbert, 2010). At the same time, the number and volume of worldwide registered and traded chemical substances has also increased. There is no doubt that developing brain is particularly vulnerable to damage by chemicals (Rice and Barone, 2000) and evaluation of chemicals for developmental neurotoxicity is critical to human health (Grandjean and Landrigan, 2006, 2014). However, only a very small number of chemicals has been tested for developmental toxicity in recent years (Middaugh et al., 2003; Makris et al., 2009), presumably because the current guidelines for DNT testing exclusively involve animal experiments (OECD, 1997, 2007) that are of poor reproducibility and predictive quality, low in throughput, prohibitively expensive and limited with regard to mechanistic insights into the toxicant's mode of action (Smirnova et al., 2014).

DNT testing is conducted for identification of chemical-induced adverse changes in the structure and function of the developing central nervous system. At present, NT and DNT testing is only officially acknowledged by regulatory authorities when done with standardized in vivo animal test methods and when conducted according to guidelines provided by the Organization for Economic Cooperation and Development (OECD). For example, NT testing involves daily oral dosing of rats for acute, sub chronic or chronic assessments for 28 days, 90 days, 1 year or longer (OECD, 1997). Primary observations include behavioral assessments and evaluation of nervous system histopathology. DNT testing evaluates in utero and early postnatal effects by daily dosing of at least 60 pregnant rats from implantation through lactation. Offspring are evaluated for neurologic and behavioral abnormalities and brain weights and neuropathology are assessed at different times through adulthood (OECD, 2007). The type of exposure (single or repeated dose) and the outcome (lethal or nonlethal; immediate or delayed effects) will result in different classifications for substances under the Globally Harmonized System (GHS).

Since there are various methods available for toxicological profiling of GABA_ARs and GlyRs (Gilbert et al., 2009a,b,d; Talwar et al., 2013) these receptors can serve as valuable molecular targets for in vitro developmental neurotoxicity testing (DNT) and provide mechanistic insights into the neurotoxicants or developmental neurotoxicants mode of action.

However, systematic screening for potentiating or inhibiting modulators of GABA_ARs and GlyRs in the context of drug development and NT/DNT testing is hampered due to lack of appropriate in vitro models. Recombinant expression systems using e.g., human embryonal kidney-derived (HEK293) cells allow systematic large scale screening for GABA_AR and GlyR modulators in high throughput format (Kruger et al., 2005; Gilbert et al., 2009a,b,d; Talwar et al., 2013; Walzik et al., 2015). Despite recombinant models being successful in the identification of GlyR chloride channel modulators (Balansa et al., 2010, 2013a,b), these systems lack of fundamental neuronal genetic programs and cell intrinsic regulators influencing the functional properties of mature neurons in vivo and are restricted to physiological, pharmacological and toxicological analysis of individual GABA_ARs and GlyRs isoforms in isolation. Modulators identified or investigated using recombinant expression systems have been reported to yield contradictive results comparing recombinant systems and native neurons. For example, NV-31 an analog of bilobalide, a major bioactive component of Ginkgo biloba herbal extracts, has been reported to inhibit recombinant GlyRs but to potentiate native hippocampal neuron GlyRs (Lynch and Chen, 2008). Hence, screening data generated using recombinant expression system may be only partially relevant to GABA_ARs and GlyRs expressed in vivo and always require time and resource intensive retesting using secondary and individual approaches. Terminally differentiated neuronal cells of human origin, e.g., primary cells from biopsy samples are rarely available, enable only a limited number of experiments and are typically derived from pathogenic tissue, rendering these cells unsuitable to systematic large-scale screening for modulators of GABA_ARs and GlyRs. Neuronal cells of animal origin such as mouse or rat are widely used for studying mammalian inhibitory neurotransmission in general and the physiological properties of GABA_ARs and GlyRs in particular but are not optimal for identification of human-specific therapeutic leads or pharmacological probes as well as for GABA_AR and GlyR mediated neurotoxicity or developmental neurotoxicity as the physiology of animals may strongly differ from human physiology. Stem cells including induced pluripotent stem cell (iPSC) and pluripotent embryonal carcinoma cells provide an enormous potential for both GABA_AR- and GlyR-targeted drug development and NT/DNT testing as they allow standardized high-throughput in vitro screening of a large number of chemicals in maturing and adult human neurons, that is time, cost and resource-effective.

Human pluripotent NTERA-2 (NT2 or TERA2.cl.SP12) stem cells are increasingly considered as a suitable model for in vitro NT and DNT studies (Couillard-Despres et al., 2008; Hill et al., 2008; Laurenza et al., 2013; Pallocca et al., 2013; Stern et al., 2014). Upon exposure to retinoic acid, the cells undergo neuronal differentiation, i.e., mimic the process of differentiation in the developing brain, and are potentially suitable to NT/DNT testing at different developmental stages ranging from non-differentiated stem cells, committed neural progenitors to differentiated neuronal, so called NT2-N cells, and glial cells (Lee and Andrews, 1986; Pleasure et al., 1992; Sandhu et al., 2002; Stewart et al., 2003; Ozdener, 2007; Coyne et al., 2011). Electrophysiological studies and extracellular recordings with NT2-N cells have demonstrated voltage-activated calcium, TTX-sensitive sodium and potassium currents, spontaneous synaptic currents as well as glutamate, N-methyl-D-aspartate (NMDA), GABA and strychnine-sensitive glycine-induced currents (Pleasure et al., 1992; Munir et al., 1996; Neelands et al., 1998; Gao et al., 2004; Coyne et al., 2011; Laurenza et al., 2013) demonstrating that these cells exhibit properties similar to those described in native human neurons thus, making them an excellent experimental model for both GABA_AR- and GlyR-targeted drug development and NT/DNT testing. Also, a variety of neuronal markers has been reported to be expressed in differentiated NT2-N cells, including β-tubulin type III, MAP-2 and synapsin I (Pleasure et al., 1992; Stewart et al., 2003; Hsu et al., 2014; Saporta et al., 2014; Stern et al., 2014).

To address the limitations of conventional in vitro models for GABA_AR- and GlyR-targeted drug, NT/DNT screening as well as of animal-based in vivo NT/DNT testing approaches described above, we aimed to establish a cell line stably expressing YFP-I152L under the control of the human ubiquitin promoter C. The promoter has been reported to drive selective protein expression in principal neurons in the mammalian brain (Wilhelm et al., 2011). NT2 cells have previously been reported to provide a suitable system for expressing exogenous proteins in terminally differentiated neurons (Pleasure et al., 1992).

YFP-I152L, an engineered variant of yellow fluorescent protein (YFP) with greatly enhanced anion sensitivity, is quenched by small anions and is thus suited to reporting anionic influx into cells (Galietta et al., 2001). The fluorescent protein has been successfully applied for structure-function analysis and compound screening with many different chloride channel types (Kruger et al., 2005; Gilbert et al., 2009a,b,d; Balansa et al., 2010, 2013a,b; Chung et al., 2010; Gebhardt et al., 2010; Talwar et al., 2013; Walzik et al., 2015).

We further aimed to apply the cell line in concentration-response experiments with GABA and glycine as well as with a selection of chemicals with known toxicity profiles on GABA_ARs and GlyRs and to compare GABA and glycine EC₅₀ and drug IC₅₀ values with published electrophysiological data and data from fluorescence based functional imaging.

To evaluate the suitability of the in vitro model to systematic large-scale functional screening in the context of GABA_AR- and GlyR-targeted drug development and NT/DNT testing, we intended to quantify the proportion of GABA_AR- and GlyR-positive cells.

Our in vitro model and methodological approach will be applicable within a framework of various individual strategies assessing NT/DNT at different stages during neuronal differentiation as well as to systematic large-scale in vitro neurophysiological, -pharmacological and -toxicological screening with GABA_ARs and GlyRs that is time, cost and resource-effective. Furthermore, in the context of in vitro-based experimental and analytical approaches for NT/DNT prediction, our methodology can contribute to reduce or even replace animal experiments and to further promote the concept of the 3Rs in biomedicine (Russell and Burch, 1959).

We have established a recombinant human pluripotent stem cell line, stably expressing halide-sensitive YFP-I152L under the control of the human ubiquitin C promoter. The cell line allows fluorescence-based GABA_AR and GlyR-targeted drug screening and in vitro NT/DNT testing at different stages of neuronal maturation. The workflow of cell line generation, stem cell differentiation and functional imaging is shown in Figure 1. Images of recombinant non-differentiated NT2-YFP-I152L stem cells and differentiated NT2-N-YFP-I152L cells as well as the principle of functional imaging using the cells are shown in Figure 2.

For the sake of feasibility, throughput and cost reasons, GABA_AR and GlyR-targeted screening is typically conducted in non-neuronal recombinant expression systems assessing individual GABA_ARs and GlyRs isoforms in isolation. Such expression systems usually also lack fundamental neuronal genetic programs and cell intrinsic regulators of mature neurons in vivo. The initial benefit is often compromised by subsequent time-consuming and cost-intensive re-screens for validation and specificity-evaluation of potential GABA_AR and GlyR-modulators. In the context of neurotoxicity (NT) or developmental neurotoxicity (DNT) testing, GABA_ARs and GlyRs can serve as molecular targets and can contribute to deciphering the molecular mechanisms of toxic effects. However, so far there are no suitable in vitro models available which comprehensively reflect the complex situation of the central nervous system in vivo and NT/DNT testing is still solely based on animal experimentation that is morally questionable, low in throughput, expensive and of rather poor predictive quality. To address these issues, we have established a recombinant human pluripotent stem cell line, stably expressing YFP-I152L that is advantageous for GABA_AR and GlyR-targeted screening in the context of lead identification and drug development as well as for NT/DNT testing for several reasons. First, the recombinant cell line allows functional profiling of GABA_ARs and GlyRs in neuronal environment, providing fundamental neuronal genetic programs and cell intrinsic regulators critical to reflecting the functional properties of mature neurons in vivo. Second, the in vitro model is of human origin, allowing for GABA_AR and GlyR-targeted screening relevant to human physiology, including identification of leads for therapeutic intervention and toxicity evaluation. Third, as the cell line used in this study mimics brain maturation to a certain extent, it allows analysis of the physiological properties of GABA_AR and GlyR during development of the central nervous system. Fourth, the cell model has been applied over a large range of passages—up to passage number 50 until now—without noticeable loss in YFP-I152L fluorescence intensity, further highlighting its applicability to standardized in vitro NT/DNT testing. Finally, differentiation of NT2 cells can be conducted in large scale format allowing for systematic high-throughput experimentation.

We exposed the cell line to GABA or glycine and demonstrated that the in vitro model is suitable to functional imaging of both GABA_ARs and GlyRs. Despite the fact that Hill coefficients compare well with data from the literature, the calculated half-maximal activation concentration for GABA and glycine is overall smaller compared to EC₅₀ concentrations previously measured in NT2-N cells using whole cell patch clamp electrophysiology (Neelands et al., 1998; Gao et al., 2004; Gao and Greenfield, 2005; Coyne et al., 2011). Although it is difficult to isolate the reason for this without directly comparing concentration-response data from the same cells generated with different methodologies, the apparent high GABA and glycine sensitivities may have been caused by the relatively long incubation time in agonist solution, i.e., the time between application of the agonist and quantification of the fluorescence intensity (30 s). Furthermore, we have previously observed differences in glycine EC₅₀, i.e., increased sensitivity for glycine, in recombinantly expressed α1, α2, and α3 GlyR, assessed with the fluorescence-based assay vs. patch clamp electrophysiology (Talwar et al., 2013).

We used the recombinant in vitro model in a case study with the drugs strychnine, picrotoxin, fipronil, lindane, bicuculline and zinc. The imaging data indicate that YFP-I152L-expressing NT2-N cells are applicable to GABA_AR and GlyR-targeted toxicological profiling and identification of ion channel-specific drugs. The drug strychnine specifically inhibited glycine but not GABA-induced anion-influx with average IC₅₀ values that correspond very well with results measured by either electrophysiological or equilibrium [³H]-strychnine displacement studies using recombinantly expressed homomeric wildtype α1 GlyRs and variants (Lynch et al., 1997; Vafa et al., 1999). Also, concentration-response experiments with pictrotoxin, a standard pharmacological tool for identifying the presence of β-subunits in recombinant and native GlyRs (Lynch, 2004) revealed half-maximal inhibition concentrations and slope values for glycine and GABA-induced anion influxes, that correspond well with results measured by patch clamp electrophysiology in recombinantly expressed homomeric a2 GlyR (Wang et al., 2007) as well as in human pluripotent stem cell-derived neurons (James et al., 2014), respectively. The alkaloid is known to strongly inhibit GABA_ARs at low micromolar concentrations, whereas GlyRs in vivo are considered much less sensitive (Lynch, 2004). However, we observed a reversed sensitivity sequence with a picrotoxin-sensitivity of GlyR that is approximately 5-fold higher compared to GABA_ARs. As it has been reported that αβ-heteromeric GlyRs were less sensitive to picrotoxin-inhibition than were α-homomeric GlyRs (Pribilla et al., 1992) we speculate that NT2-N-YFP-I152L cells predominantly express α-homomeric GlyR rather than αβ-heteromeric GlyRs, although this is no explanation for the reversed picrotoxin-sensitivities of GlyRs and GABA_ARs. Bicuculline, a purported selective antagonist of the GABA_A receptor, inhibited the GABA-induced fluorescence quench with a half-maximal inhibition concentration similar to values revealed from rat primary neurons by whole cell electrophysiological recordings (Kumamoto and Murata, 1995). Bicuculline also inhibited glycine induced I⁻ influx in NT2-N-YFP-I152L cells with about 10-fold lower sensitivity compared to GABA-dependent anion influx, highlighting its specificity to GABA_AR on the one hand, but also confirming previous reports on its inhibitory action assessed in both recombinantly expressed (Sun and Machu, 2000) and native glycine receptors (Bhattarai et al., 2016) on the other hand. The IC₅₀ value for glycine-induced fluorescence quench revealed in presence of increasing concentration of the insecticide lindane is about one order of magnitude smaller compared to electrophysiological recordings from recombinantly expressed GlyRs (Islam and Lynch, 2012). These data suggest that NT2-N-YFP-I152L cells predominantly express homomeric GlyRs as lindane has proven to selectively inhibit homomeric but not heteromeric GlyRs (Islam and Lynch, 2012; Talwar et al., 2013). This conclusion is further supported by the above mentioned high picrotoxin-sensitivity of GlyRs. Surprisingly, lindane showed no dose-dependent inhibitory activity on GABA-induced anion influx, although lindane-sensitivity of GABA_ARs has been reported by many studies conducted in various culture models and cell types (Narahashi, 1996; Ogata et al., 1988; Vale et al., 2003). A study conducted by Belelli et al. (1999) demonstrated no effect on GABA-evoked currents mediated by anesthetic-insensitive wild-type GABA receptors composed of the (rho 1) subunit (Belelli et al., 1999), suggesting expression of GABA_A rho 1 receptor in the examined NT2-N-YFP-I152L cells. Indeed there are a number of studies indicating profound changes in subunit expression and pharmacology during neuronal differentiation—similar to developmental changes in GABA_AR occurring in neurons of the developing central nervous system—and demonstrating a large variety of neurotransmission phenotypes of non-differentiated NT2 and differentiated NT2-N cell in vitro (Neelands et al., 1998, 1999; Guillemain et al., 2000). This question however was not further evaluated in the present study. Fipronil, a broad-spectrum insecticide, inhibited both GABA and glycine induced anion influx in NT2-N cells with sensitivities that correspond very well to results measured by patch clamp electrophysiology in recombinantly expressed GlyRs and GABA_AR (Ratra et al., 2001; Li and Akk, 2008; Islam and Lynch, 2012). Zinc is widely recognized as a modulator of both, GABAAR and GlyR (Smart et al., 1994). For GlyRs, potentiation as well as inhibition of glycine-activated currents by low (< 10 μM) and high concentrations (>100 μM) of zinc, respectively has been reported (Harvey et al., 1999). For GABAARs, inhibition has been shown at concentrations >10 μM (Kumamoto and Murata, 1995). Although we observed a minor increase of a glycine-induced fluorescence quench (Approximately 10%) in presence of 1 and 10 μM zinc, this effect is not significant. In contrast, zinc-dependent inhibition was observed at concentrations >100 μM for both GABA and glycine-induced anion influx, confirming previous reports on zinc-sensitive GABA-induced currents recorded from NT2-N cells (Gao et al., 2004). The zinc IC₅₀ could not be calculated due to a too small range of applied concentrations but presumably exceeds 1 mM, suggesting the presence of γ subunits which have shown to confer zinc insensitivity to αβ GABA_ARs (Smart et al., 1991).

We quantified the proportion of GABA_AR and GlyR positive NT2-N-YFP-I152L cells based on images of the cells and we identified a percentage of approximately 20% of cells expressing GABA_ARs and GlyRs, respectively. Despite the fact that the proportion of glycine and GABA sensitive cells are comparable, it cannot be concluded from our data whether the cells are simultaneously sensitive to both glycine and GABA or respond to either of the neurotransmitters. Although the calculated proportions have proven stable between different batches of differentiation cultures, the total percentage of GABA_AR and GlyR positive NT2-N-YFP-I152L cells is not optimal for plate reader, i.e., photomultiplier or cell population-based screening, that typically requires a high number of reacting, e.g., GlyR or GABA_AR-positive cells and allows experimentation in high-throughput screening mode. If desired or required, the proportion of NT2-N-YFP-I152L cells may be increased by size-exclusion filtering using e.g., a simple nylon filter of defined mesh size or even more sophisticated cell sorting approaches. As highlighted by gray and white arrows in the fluorescence micrograph depicted in Figure 6A and the histogram shown in Figure 6D, NT2-N cells are much smaller compared to non-neuronal cells, presumably facilitating size-dependent population-increase of NT2-N cells. Despite the fact that plate reader-based screening allows experimentation in high or even ultra-high-screening mode it is disadvantageous with regard to cellular heterogeneity. Cultures of NT2 cells have been reported to express a large variety of non-neuronal and neuronal phenotypes (Guillemain et al., 2000), as we have shown in the case study using drugs with known and partly GABA_AR and GlyR-specific pharmacological and toxicological profiles. Besides their neuronal properties and human origin, the heterogeneity of NT2-N cell cultures is probably one of the most important advantages over homogeneous, e.g., recombinant expression systems, as it reflects the in vivo situation to a much higher extent than simplifying in vitro models. Thus, microscopy-based high-content imaging of NT2-N cells, evaluating the physiological pharmacological and toxicological properties at single cell level, and in the context of cellular heterogeneity, is likely to be the optimal approach for systematic GABA_AR and GlyR-targeted drug screening as well as NT/DNT testing.

To confirm the validity of the morphology or size-based selection criterion, we conducted functional imaging experiments with non-treated NT2-YFP-I152L stem cells and quantified the average cell size as well as the percentage of stem cells, responding with at least 20% fluorescence quench upon exposure to saturating GABA or glycine concentration, respectively (see Supplementary Figure S1). The average cell size (pixel), calculated from two individual experiments each (see Supplementary Figure S1C) is comparable between experiments (GABA, replicate #1: 397 ± 254 pixel, n = 650 cells, replicate #2: 385 ± 252 pixel, n = 547 cells; glycine, replicate #1: 403 ± 243 pixel, n = 589, replicate #2: 400 ± 256 pixel, n = 654) and differs from the size of small and large cells, respectively, as indicated in Figure 6D. The percentage of non-treated NT2-YFP-I152L stem cells, responding with at least 20% fluorescence quench upon exposure to 1 mM saturating GABA (replicate #1: 0.62%, replicate #2: 0.37%) or glycine (replicate #1: 0.68%, replicate #2: 0.92%) concentration, respectively (see Supplementary Figure S1D), also differs considerably from data obtained with retinoic acid-exposed NT2-N-YFP-I152L cells. These data clearly demonstrate that the morphology, e.g., the size, as well as the functional properties, i.e., the sensitivity to GABA or glycine, vary between non-treated stem cells and NT2-YFP-I152L differentiation cultures containing small neuronal and large non-neuronal cells and confirming the validity of the employed size-based selection criterion.

We have previously published a method allowing for assessment of ligand-gated ion channels in the context of cellular heterogeneity that is based on progressive receptor activation and iterative fluorescence imaging (Talwar et al., 2013). This method could easily be adapted for application with the established in vitro model and has the power to deliver imaging data of unsurpassed functional content presumably outranging any other existing technique for assessing physiological properties of ion channels with regard to throughput.

Due to the fact that our method is based on microscopic evaluation using a single fluorescence indicator, it allows additional fluorescence or luminescence-based markers to be implemented for multiplexing, such as ion or pH-sensitive probes e.g., for parallel analysis of further ion channels or transporters, to assess the activity of intracellular, e.g., toxicity-relevant signaling pathways, or for mapping cellular morphology to functional phenotypes.

It is important to mention that the applicability of the cell line as well as the experimental approach depends on the proteins to be evaluated and targeted, on the individual experimental setup, the available instrumental infrastructure and the biological question to be assessed. While this work focuses on functional profiling and GABA_AR and GlyR-targeted drug screening as well as neurotoxicity and developmental neurotoxicity, our in vitro model and methodological approach could also be adapted for other proteins and strategies, such as RNAi or combined compound and RNAi screening, for single-endpoint or time-resolved functional expression analysis or for approaches using overexpression libraries. Altogether, this work contributes to furthering the applicability of cell-based high-throughput functional screening and provides a means for large-scale characterization of neuronal proteins in the context of in vitro-based NT/DNT prediction thus, promoting a systems-level understanding of human physiology in homeostasis and disease.

KK, OF, and DG conceived the project. KK conducted experiments. KK and DG analyzed the data. KK and DG wrote the paper. All authors commented and agreed on the manuscript.