10) where R(pCa) represents the column of the measured ratios, and
20. Do not repeat the recording in a different field of view in the same culture even after a washout of concanamycin A and a
To account for systematic errors such as variance in cultures, choice of field of view, pipetting errors, and temperature, the results should be trusted only if based on a sufficient sample size of at least ten different coverslips per experimental condition.
Be sure to double-check statistical power of your results [29].
Acknowledgments
This work was supported by Else-Krửner- Fresenius Stiftung grant 2012_A35.
Supplementary Files
Exemplary MATLAB code for processing a vesicle pool size record- ing can be found in the GitHub repository available at https://
github.com/janawrosch/VesiclePoolSizes.
References
1. Ryan TA, Smith SJ (1995) Vesicle pool mobi- lization during action potential firing at hip- pocampal synapses. Neuron 14(5):983–989 2. Wienisch M, Klingauf J (2006) Vesicular pro-
teins exocytosed and subsequently retrieved by compensatory endocytosis are nonidentical.
Nat Neurosci 9(8):1019–1027
3. Atwood HL, Karunanithi S (2002) Diversification of synaptic strength: presynap- tic elements. Nat Rev Neurosci 3(7):497–516 4. Wang X, Pinter MJ, Rich MM (2016)
Reversible recruitment of a homeostatic reserve pool of synaptic vesicles underlies rapid homeostatic plasticity of quantal content.
J Neurosci 36(3):828–836
5. Waters J, Smith SJ (2002) Vesicle pool parti- tioning influences presynaptic diversity and weighting in rat hippocampal synapses.
J Physiol 541(Pt 3):811–823
6. Rosenmund C, Stevens CF (1996) Definition of the readily releasable pool of vesicles at hip-
pocampal synapses. Neuron 16(6):1197–1207
7. Wilhelm BG, Groemer TW, Rizzoli SO (2010) The same synaptic vesicles drive active and spontaneous release. Nat Neurosci 13(12):1454–1456
8. Schikorski T, Stevens CF (2001) Morphological correlates of functionally defined synaptic vesi- cle populations. Nat Neurosci 4(4):391–395
204
9. Jung J, Loy K, Schilling EM et al (2014) The antidepressant fluoxetine mobilizes vesicles to the recycling pool of rat hippocampal synapses during high activity. Mol Neurobiol 49(2):916–930
10. Tagliatti E, Fadda M, Falace A et al (2016) Arf6 regulates the cycling and the readily releasable pool of synaptic vesicles at hippo- campal synapse. Elife 5:e10116
11. Welzel O, Tischbirek CH, Jung J et al (2010) Synapse clusters are preferentially formed by synapses with large recycling pool sizes. PLoS One 5(10):e13514
12. Ryan TA, Li L, Chin LS et al (1996) Synaptic vesicle recycling in synapsin I knock-out mice.
J Cell Biol 134(5):1219–1227
13. Marra V, Burden JJ, Crawford F et al (2014) Ultrastructural readout of functional synaptic vesicle pools in hippocampal slices based on FM dye labeling and photoconversion. Nat Protoc 9(6):1337–1347
14. Welzel O, Henkel AW, Stroebel AM et al (2011) Systematic heterogeneity of fractional vesicle pool sizes and release rates of hippo- campal synapses. Biophys J 100(3):593–601 15. Miesenbock G, De Angelis DA, Rothman JE
(1998) Visualizing secretion and synaptic transmission with pH-sensitive green fluores- cent proteins. Nature 394(6689):192–195 16. Hua Y, Sinha R, Thiel CS et al (2011) A read-
ily retrievable pool of synaptic vesicles. Nat Neurosci 14(7):833–839
17. Sankaranarayanan S, De Angelis D, Rothman JE et al (2000) The use of pHluorins for opti- cal measurements of presynaptic activity.
Biophys J 79(4):2199–2208
18. Rother M, Brauner JM, Ebert K et al (2014) Dynamic properties of the alkaline vesicle pop- ulation at hippocampal synapses. PLoS One 9(7):e102723
19. Li Z, Burrone J, Tyler WJ et al (2005) Synaptic vesicle recycling studied in transgenic mice expressing synaptopHluorin. Proc Natl Acad Sci U S A 102(17):6131–6136
20. Groemer TW, Klingauf J (2007) Synaptic vesi- cles recycling spontaneously and during activ- ity belong to the same vesicle pool. Nat Neurosci 10(2):145–147
21. Sbalzarini IF, Koumoutsakos P (2005) Feature point tracking and trajectory analysis for video imaging in cell biology. J Struct Biol 151(2):182–195
22. Kaech S, Banker G (2006) Culturing hippo- campal neurons. Nat Protoc 1(5):2406–2415 23. Takahashi K, Tanabe K, Ohnuki M et al (2007)
Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131(5):861–872
24. Threadgill R, Bobb K, Ghosh A (1997) Regulation of dendritic growth and remodel- ing by Rho, Rac, and Cdc42. Neuron 19(3):625–634
25. Transfection of mammalian cells by electro- poration (2006) Nat Method. 3(1):67–68 26. Zhang XS, Huang J, Zhan CQ et al (2016)
Different influences of lipofection and electro- transfection on in vitro gene delivery to pri- mary cultured cortex neurons. Pain Physician 19(3):189–196
27. Royle SJ, Granseth B, Odermatt B et al (2008) Imaging phluorin-based probes at hippocam- pal synapses. Methods Mol Biol 457:293–303 28. Jia H, Rochefort NL, Chen X et al (2011) In
vivo two-photon imaging of sensory-evoked dendritic calcium signals in cortical neurons.
Nat Protoc 6(1):28–35
29. Button KS, Ioannidis JP, Mokrysz C et al (2013) Power failure: why small sample size undermines the reliability of neuroscience. Nat Rev Neurosci 14(5):365–376
Jana K. Wrosch and Teja W. Groemer
205
Chapter 16
Phenotyping Cellular Viability by Functional Analysis of Ion Channels: GlyR-Targeted Screening in NT2-N Cells
Katharina Kuenzel*, Sepideh Abolpour Mofrad*, and Daniel F. Gilbert
Abstract
Glycine receptor chloride channels (GlyRs) are attractive drug targets for therapeutic intervention and are also more and more recognized in the context of in vitro neurotoxicity and developmental neurotoxicity testing. Assaying the functional properties of GlyR can serve as an indicator of cellular viability and the integrity of the developing and mature central nervous system. Human pluripotent NTERA-2 (NT2) stem cells undergo neuronal differentiation upon stimulation with retinoic acid and express a large variety of neuronal proteins—including GlyR. YFP-I152L, a halide-sensitive variant of yellow fluorescent protein, allows high-throughput fluorescence-based functional analysis of GlyRs in NT2 cells. Here we describe a protocol for phenotyping of cellular viability by functional analysis of GlyR in neuronally differentiated NT2 (NT2-N) cells using YFP-I152L as a reporter of functional integrity of GlyRs. The protocol describes neuronal differentiation of NT2 stem cells, transient transfection of NT2-N cells with YFP-I152L as well as functional imaging and analysis of data from high-content imaging.
Key words Human pluripotent embryonal teratocarcinoma stem cells, NT2 cells, NT2-N cells, Glycine receptor chloride channel (GlyR), YFP-I152L, Cell viability, Toxicological screening
1 Introduction
Glycine receptors (GlyRs) are ligand-gated ion channels which mediate inhibitory neurotransmission in the central nervous system (CNS). In mature neurons, upon activation by the amino acid and neurotransmitter glycine, GlyRs conduct a hyperpolarizing inward- directed anion current into the cells [1–3]. Impaired channel func- tion, by, e.g., genetic or molecular perturbation, can lead to severe neurological disorders including epilepsy [4–6], neuropathic pain [7], chronic pain sensitization [2, 8, 9], and hyperekplexia [10, 11]
and has also been associated with neurotoxicity [12–15]. Due to their important role in inhibitory neurotransmission, these channels are considered attractive drug targets for therapeutic intervention
*Katharina Kuenzel and Sepideh Abolpour Mofrad contributed equally to this work.
Daniel F. Gilbert and Oliver Friedrich (eds.), Cell Viability Assays: Methods and Protocols, Methods in Molecular Biology, vol. 1601, DOI 10.1007/978-1-4939-6960-9_16, © Springer Science+Business Media LLC 2017
206
[16] and are also increasingly recognized in the context of in vitro neurotoxicity (NT) [12, 13, 17–19] and developmental neurotox- icity (DNT) testing [14, 15]. Assaying the functional properties of GlyR can serve as an indicator of cellular viability and the integrity of the developing and mature central nervous system.
Human pluripotent NTERA-2 (NT2 or TERA2.cl.SP12) stem cells undergo neuronal differentiation upon exposure to retinoic acid and mimic the process of differentiation in the developing brain including developmental stages ranging from nondifferenti- ated stem cells, committed neural progenitors to differentiated neuronal—so-called NT2-N cells—and glial cells [20–25].
Electrophysiological studies 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 [14, 21, 25–29], indicating that these cells exhibit properties similar to those described in native human neurons.
YFP-I152L, a variant of yellow fluorescent protein (YFP) with strongly enhanced anion sensitivity, is quenched by small anions and is thus suitable to reporting anionic influx into cells [30]. Figure 1 shows the principle of cellular viability phenotyp- ing by functional analysis of GlyRs using the fluorescence reporter YFP- I152L. The fluorescent protein has been success- fully and repeatedly applied for compound screening and struc- ture-function analysis with many different chloride channel types [14, 15, 31–39].
Here we describe a protocol for phenotyping of cellular viabil- ity by functional analysis of GlyRs in NT2 cells using recombi- nantly expressed YFP-I152L as a reporter of GlyR activation.
Fig. 1 Principle of cell viability phenotyping by functional analysis of GlyRs using the fluorescence reporter YFP-I152L. (a) Fluorescence intensity of YFP-I152L is strong in resting cells. (b) Upon activation by its ligand, GlyRs conduct an inward-directed anion current, leading to fluorescence quenching of YFP-I152L
Katharina Kuenzel et al.
YFP-I152L is expressed 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 [40]. NT2 cells have previously been reported to provide a suitable system for expressing exogenous proteins in terminally differenti- ated neurons [21]. A timeline of neuronal differentiation of NT2 cells in monolayer cultures previously developed in our laboratory [41] and the experimental workflow are shown in Fig. 2.
2 Materials
1. Human pluripotent teratocarcinoma cells (NTERA-2.cl.D1, CRL-1973™, ATCC).