{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,11,1]],"date-time":"2025-11-01T13:47:15Z","timestamp":1762004835650},"reference-count":73,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2016,9,13]],"date-time":"2016-09-13T00:00:00Z","timestamp":1473724800000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2016,9,13]],"date-time":"2016-09-13T00:00:00Z","timestamp":1473724800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Sci Rep"],"abstract":"<jats:title>Abstract<\/jats:title><jats:p>The generation of human neural tissue-like 3D structures holds great promise for disease modeling, drug discovery and regenerative medicine strategies. Promoting the establishment of complex cell-cell interactions, 3D culture systems enable the development of human cell-based models with increased physiological relevance, over monolayer cultures. Here, we demonstrate the establishment of neuronal and astrocytic metabolic signatures and shuttles in a human 3D neural cell model, namely the glutamine-glutamate-GABA shuttle. This was indicated by labeling of neuronal GABA following incubation with the glia-specific substrate [2-<jats:sup>13<\/jats:sup>C]acetate, which decreased by methionine sulfoximine-induced inhibition of the glial enzyme glutamine synthetase. Cell metabolic specialization was further demonstrated by higher pyruvate carboxylase-derived labeling in glutamine than in glutamate, indicating its activity in astrocytes and not in neurons. Exposure to the neurotoxin acrylamide resulted in intracellular accumulation of glutamate and decreased GABA synthesis. These results suggest an acrylamide-induced impairment of neuronal synaptic vesicle trafficking and imbalanced glutamine-glutamate-GABA cycle, due to loss of cell-cell contacts at synaptic sites. This work demonstrates, for the first time to our knowledge, that neural differentiation of human cells in a 3D setting recapitulates neuronal-astrocytic metabolic interactions, highlighting the relevance of these models for toxicology and better understanding the crosstalk between human neural cells.<\/jats:p>","DOI":"10.1038\/srep33285","type":"journal-article","created":{"date-parts":[[2016,9,13]],"date-time":"2016-09-13T10:14:54Z","timestamp":1473761694000},"update-policy":"http:\/\/dx.doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":15,"title":["Functional metabolic interactions of human neuron-astrocyte 3D in vitro networks"],"prefix":"10.1038","volume":"6","author":[{"given":"Daniel","family":"Sim\u00e3o","sequence":"first","affiliation":[]},{"given":"Ana P.","family":"Terrasso","sequence":"additional","affiliation":[]},{"given":"Ana P.","family":"Teixeira","sequence":"additional","affiliation":[]},{"given":"Catarina","family":"Brito","sequence":"additional","affiliation":[]},{"given":"Ursula","family":"Sonnewald","sequence":"additional","affiliation":[]},{"given":"Paula M.","family":"Alves","sequence":"additional","affiliation":[]}],"member":"297","published-online":{"date-parts":[[2016,9,13]]},"reference":[{"key":"BFsrep33285_CR1","doi-asserted-by":"publisher","first-page":"37","DOI":"10.1016\/j.drudis.2014.10.011","volume":"20","author":"S Giri","year":"2014","unstructured":"Giri, S. & Bader, A. A low-cost, high-quality new drug discovery process using patient-derived induced pluripotent stem cells. Drug Discov. Today 20, 37\u201349 (2014).","journal-title":"Drug Discov. Today"},{"key":"BFsrep33285_CR2","doi-asserted-by":"publisher","first-page":"203","DOI":"10.1038\/nrd3078","volume":"9","author":"SM Paul","year":"2010","unstructured":"Paul, S. M. et al. How to improve R&D productivity: the pharmaceutical industry\u2019s grand challenge. Nat. Rev. Drug Discov. 9, 203\u2013214 (2010).","journal-title":"Nat. Rev. Drug Discov."},{"key":"BFsrep33285_CR3","doi-asserted-by":"publisher","first-page":"85","DOI":"10.1038\/nrd4239","volume":"13","author":"A Mullard","year":"2014","unstructured":"Mullard, A. 2013 FDA drug approvals. Nat. Rev. Drug Discov. 13, 85\u201389 (2014).","journal-title":"Nat. Rev. Drug Discov."},{"key":"BFsrep33285_CR4","doi-asserted-by":"publisher","first-page":"161","DOI":"10.1038\/nrd4570","volume":"14","author":"D Calcoen","year":"2015","unstructured":"Calcoen, D., Elias, L. & Yu, X. What does it take to produce a breakthrough drug? Nat. Rev. Drug Discov. 14, 161\u2013162 (2015).","journal-title":"Nat. Rev. Drug Discov."},{"key":"BFsrep33285_CR5","doi-asserted-by":"publisher","first-page":"1210","DOI":"10.1038\/nm.2224","volume":"16","author":"M Jucker","year":"2010","unstructured":"Jucker, M. The benefits and limitations of animal models for translational research in neurodegenerative diseases. Nat. Med. 16, 1210\u20131214 (2010).","journal-title":"Nat. Med."},{"key":"BFsrep33285_CR6","doi-asserted-by":"publisher","first-page":"531","DOI":"10.1038\/483531a","volume":"483","author":"CG Begley","year":"2012","unstructured":"Begley, C. G. & Ellis, L. M. Drug development: Raise standards for preclinical cancer research. Nature 483, 531\u2013533 (2012).","journal-title":"Nature"},{"key":"BFsrep33285_CR7","first-page":"114","volume":"6","author":"IW Mak","year":"2014","unstructured":"Mak, I. W., Evaniew, N. & Ghert, M. Lost in translation: animal models and clinical trials in cancer treatment. Am. J. Transl. Res. 6, 114\u2013118 (2014).","journal-title":"Am. J. Transl. Res."},{"key":"BFsrep33285_CR8","doi-asserted-by":"publisher","first-page":"1043","DOI":"10.1016\/j.bbadis.2009.08.014","volume":"1792","author":"B Sch\u00fcle","year":"2009","unstructured":"Sch\u00fcle, B., Pera, R. a R. & Langston, J. W. Can cellular models revolutionize drug discovery in Parkinson\u2019s disease? Biochim. Biophys. Acta 1792, 1043\u20131051 (2009).","journal-title":"Biochim. Biophys. Acta"},{"key":"BFsrep33285_CR9","doi-asserted-by":"publisher","first-page":"285","DOI":"10.1016\/0012-1606(84)90316-6","volume":"103","author":"PW Andrews","year":"1984","unstructured":"Andrews, P. W. Retinoic acid induces neuronal differentiation of a cloned human embryonal carcinoma cell line in vitro. Dev. Biol. 103, 285\u2013293 (1984).","journal-title":"Dev. Biol."},{"key":"BFsrep33285_CR10","doi-asserted-by":"publisher","first-page":"1802","DOI":"10.1523\/JNEUROSCI.12-05-01802.1992","volume":"12","author":"SJ Pleasure","year":"1992","unstructured":"Pleasure, S. J., Page, C. & Lee, V. M. Pure, postmitotic, polarized human neurons derived from NTera 2 cells provide a system for expressing exogenous proteins in terminally differentiated neurons. J. Neurosci. 12, 1802\u20131815 (1992).","journal-title":"J. Neurosci."},{"key":"BFsrep33285_CR11","doi-asserted-by":"publisher","first-page":"1685","DOI":"10.1002\/jnr.22692","volume":"89","author":"CE Goodfellow","year":"2011","unstructured":"Goodfellow, C. E., Graham, S. E., Dragunow, M. & Glass, M. Characterization of NTera2\/D1 cells as a model system for the investigation of cannabinoid function in human neurons and astrocytes. J. Neurosci. Res. 89, 1685\u20131697 (2011).","journal-title":"J. Neurosci. Res."},{"key":"BFsrep33285_CR12","doi-asserted-by":"publisher","first-page":"3521","DOI":"10.1046\/j.1460-9568.2000.00230.x","volume":"12","author":"SA Przyborski","year":"2000","unstructured":"Przyborski, S. A., Morton, I. E., Wood, A. & Andrews, P. W. Developmental regulation of neurogenesis in the pluripotent human embryonal carcinoma cell line NTERA-2. Eur. J. Neurosci. 12, 3521\u20133528 (2000).","journal-title":"Eur. J. Neurosci."},{"key":"BFsrep33285_CR13","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1002\/(SICI)1096-9861(19990428)407:1<1::AID-CNE1>3.0.CO;2-Z","volume":"407","author":"RS Hartley","year":"1999","unstructured":"Hartley, R. S., Margulis, M., Fishman, P. S., Lee, V. M. Y. & Tang, C. M. Functional synapses are formed between human NTera2 (NT2N, hNT) neurons grown on astrocytes. J. Comp. Neurol. 407, 1\u201310 (1999).","journal-title":"J. Comp. Neurol."},{"key":"BFsrep33285_CR14","doi-asserted-by":"publisher","first-page":"e16174","DOI":"10.1371\/journal.pone.0016174","volume":"6","author":"DE Coyle","year":"2011","unstructured":"Coyle, D. E., Li, J. & Baccei, M. Regional differentiation of retinoic acid-induced human pluripotent embryonic carcinoma stem cell neurons. PLoS One 6, e16174 (2011).","journal-title":"PLoS One"},{"key":"BFsrep33285_CR15","doi-asserted-by":"publisher","first-page":"82","DOI":"10.1016\/j.jbiotec.2014.12.011","volume":"205","author":"AP Terrasso","year":"2015","unstructured":"Terrasso, A. P. et al. Novel scalable 3D cell based model for in vitro neurotoxicity testing: Combining human differentiated neurospheres with gene expression and functional endpoints. J. Biotechnol. 205, 82\u201392 (2015).","journal-title":"J. Biotechnol."},{"key":"BFsrep33285_CR16","doi-asserted-by":"publisher","first-page":"243","DOI":"10.1016\/j.tox.2008.05.014","volume":"249","author":"EJ Hill","year":"2008","unstructured":"Hill, E. J., Woehrling, E. K., Prince, M. & Coleman, M. D. Differentiating human NT2\/D1 neurospheres as a versatile in vitro 3D model system for developmental neurotoxicity testing. Toxicology 249, 243\u2013250 (2008).","journal-title":"Toxicology"},{"key":"BFsrep33285_CR17","doi-asserted-by":"publisher","first-page":"679","DOI":"10.1016\/j.ijdevneu.2013.03.002","volume":"31","author":"I Laurenza","year":"2013","unstructured":"Laurenza, I. et al. A human pluripotent carcinoma stem cell-based model for in vitro developmental neurotoxicity testing: Effects of methylmercury, lead and aluminum evaluated by gene expression studies. Int. J. Dev. Neurosci. 31, 679\u2013961 (2013).","journal-title":"Int. J. Dev. Neurosci."},{"key":"BFsrep33285_CR18","doi-asserted-by":"publisher","first-page":"127","DOI":"10.1007\/s00204-013-1098-1","volume":"88","author":"M Stern","year":"2014","unstructured":"Stern, M., Gierse, A., Tan, S. & Bicker, G. Human Ntera2 cells as a predictive in vitro test system for developmental neurotoxicity. Arch. Toxicol. 88, 127\u2013136 (2014).","journal-title":"Arch. Toxicol."},{"key":"BFsrep33285_CR19","doi-asserted-by":"publisher","first-page":"839","DOI":"10.1038\/nrm2236","volume":"8","author":"F Pampaloni","year":"2007","unstructured":"Pampaloni, F., Reynaud, E. G. & Stelzer, E. H. K. The third dimension bridges the gap between cell culture and live tissue. Nat. Rev. Mol. Cell Biol. 8, 839\u2013845 (2007).","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"BFsrep33285_CR20","doi-asserted-by":"publisher","first-page":"806","DOI":"10.2741\/2721","volume":"13","author":"W Potter","year":"2008","unstructured":"Potter, W., Kalil, R. E. & Kao, W. J. Biomimetic material systems for neural progenitor cell-based therapy. Front Biosci 13, 806\u2013821 (2008).","journal-title":"Front Biosci"},{"key":"BFsrep33285_CR21","doi-asserted-by":"publisher","first-page":"1131","DOI":"10.1289\/ehp.0800207","volume":"117","author":"M Moors","year":"2009","unstructured":"Moors, M. et al. Human neurospheres as three-dimensional cellular systems for developmental neurotoxicity testing. Env. Heal. Perspect 117, 1131\u20131138 (2009).","journal-title":"Env. Heal. Perspect"},{"key":"BFsrep33285_CR22","doi-asserted-by":"publisher","first-page":"671","DOI":"10.1038\/nmeth.3415","volume":"12","author":"AM Pa\u015fca","year":"2015","unstructured":"Pa\u015fca, A. M. et al. Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture. Nat. Methods 12, 671\u2013678 (2015).","journal-title":"Nat. Methods"},{"key":"BFsrep33285_CR23","doi-asserted-by":"publisher","first-page":"654","DOI":"10.1089\/ten.tea.2014.0079","volume":"21","author":"D Sim\u00e3o","year":"2015","unstructured":"Sim\u00e3o, D. et al. Modeling human neural functionality in vitro: three-dimensional culture for dopaminergic differentiation. Tissue Eng. Part A 21, 654\u2013668 (2015).","journal-title":"Tissue Eng. Part A"},{"key":"BFsrep33285_CR24","doi-asserted-by":"publisher","first-page":"274","DOI":"10.1038\/nature13800","volume":"515","author":"S Choi","year":"2014","unstructured":"Choi, S. et al. A three-dimensional human neural cell culture model of Alzheimer\u2019s disease. Nature 515, 274\u2013278 (2014).","journal-title":"Nature"},{"key":"BFsrep33285_CR25","doi-asserted-by":"publisher","first-page":"209","DOI":"10.14573\/altex.2013.2.209","volume":"30","author":"T Ramirez","year":"2013","unstructured":"Ramirez, T. et al. Metabolomics in toxicology and preclinical research. ALTEX 30, 209\u2013225 (2013).","journal-title":"ALTEX"},{"key":"BFsrep33285_CR26","doi-asserted-by":"publisher","first-page":"417","DOI":"10.1002\/(SICI)1097-4547(19990815)57:4<417::AID-JNR1>3.0.CO;2-N","volume":"57","author":"L Hertz","year":"1999","unstructured":"Hertz, L., Dringen, R., Schousboe, A. & Robinson, S. R. Astrocytes: Glutamate producers for neurons. J. Neurosci. Res. 57, 417\u2013428 (1999).","journal-title":"J. Neurosci. Res."},{"key":"BFsrep33285_CR27","doi-asserted-by":"publisher","first-page":"1386","DOI":"10.1038\/jcbfm.2013.81","volume":"33","author":"MA Tarczyluk","year":"2013","unstructured":"Tarczyluk, M. A. et al. Functional astrocyte-neuron lactate shuttle in a human stem cell-derived neuronal network. J. Cereb. Blood Flow Metab. 33, 1386\u20131393 (2013).","journal-title":"J. Cereb. Blood Flow Metab."},{"key":"BFsrep33285_CR28","doi-asserted-by":"publisher","first-page":"4119","DOI":"10.1111\/bph.13193","volume":"172","author":"L Efremova","year":"2015","unstructured":"Efremova, L. et al. Prevention of the degeneration of human dopaminergic neurons in an astrocyte co-culture system allowing endogenous drug metabolism. Br. J. Pharmacol. 172, 4119\u20134132 (2015).","journal-title":"Br. J. Pharmacol."},{"key":"BFsrep33285_CR29","doi-asserted-by":"publisher","first-page":"2469","DOI":"10.1046\/j.1471-4159.1999.0732469.x","volume":"73","author":"KS McNaught","year":"1999","unstructured":"McNaught, K. S. & Jenner, P. Altered glial function causes neuronal death and increases neuronal susceptibility to 1-methyl-4-phenylpyridinium- and 6-hydroxydopamine-induced toxicity in astrocytic\/ventral mesencephalic co-cultures. J. Neurochem. 73, 2469\u20132476 (1999).","journal-title":"J. Neurochem."},{"key":"BFsrep33285_CR30","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1038\/ncomms1518","volume":"2","author":"P Shi","year":"2011","unstructured":"Shi, P. et al. Synapse microarray identification of small molecules that enhance synaptogenesis. Nat. Commun. 2, 1\u201310 (2011).","journal-title":"Nat. Commun."},{"key":"BFsrep33285_CR31","doi-asserted-by":"crossref","unstructured":"Giordano, G. & Costa, L. G. Morphological assessment of neurite outgrowth in hippocampal neuron-astrocyte co-cultures. Curr. Protoc. Toxicol. Chapter 11, Unit 11.16 (2012).","DOI":"10.1002\/0471140856.tx1116s52"},{"key":"BFsrep33285_CR32","doi-asserted-by":"publisher","first-page":"641","DOI":"10.1111\/j.1471-4159.2006.03913.x","volume":"98","author":"LK Bak","year":"2006","unstructured":"Bak, L. K., Schousboe, A. & Waagepetersen, H. S. The glutamate\/GABA-glutamine cycle: Aspects of transport, neurotransmitter homeostasis and ammonia transfer. J. Neurochem. 98, 641\u2013653 (2006).","journal-title":"J. Neurochem."},{"key":"BFsrep33285_CR33","doi-asserted-by":"publisher","first-page":"9","DOI":"10.3389\/fnene.2013.00009","volume":"5","author":"TB Rodrigues","year":"2013","unstructured":"Rodrigues, T. B., Valette, J. & Bouzier-Sore, A.-K. 13C NMR spectroscopy applications to brain energy metabolism. Front. Neuroenergetics 5, 9 (2013).","journal-title":"Front. Neuroenergetics"},{"key":"BFsrep33285_CR34","doi-asserted-by":"publisher","first-page":"943","DOI":"10.1002\/nbm.1772","volume":"24","author":"DL Rothman","year":"2011","unstructured":"Rothman, D. L., de Feyter, H. M., de Graaf, R. a., Mason, G. F. & Behar, K. L. 13C MRS studies of neuroenergetics and neurotransmitter cycling in humans. NMR Biomed. 24, 943\u2013957 (2011).","journal-title":"NMR Biomed."},{"key":"BFsrep33285_CR35","doi-asserted-by":"publisher","first-page":"424","DOI":"10.1002\/nbm.837","volume":"16","author":"U Sonnewald","year":"2003","unstructured":"Sonnewald, U. & Kondziella, D. Neuronal glial interaction in different neurological diseases studied by ex vivo 13C NMR spectroscopy. NMR Biomed. 16, 424\u2013429 (2003).","journal-title":"NMR Biomed."},{"key":"BFsrep33285_CR36","doi-asserted-by":"publisher","first-page":"6807","DOI":"10.1038\/ncomms7807","volume":"6","author":"I Lundgaard","year":"2015","unstructured":"Lundgaard, I. et al. Direct neuronal glucose uptake heralds activity-dependent increases in cerebral metabolism. Nat. Commun. 6, 6807 (2015).","journal-title":"Nat. Commun."},{"key":"BFsrep33285_CR37","doi-asserted-by":"publisher","first-page":"19","DOI":"10.1016\/0197-0186(93)90064-C","volume":"22","author":"U Sonnewald","year":"1993","unstructured":"Sonnewald, U. et al. Direct demonstration by [13C]NMR spectroscopy that glutamine from astrocytes is a precursor for GABA synthesis in neurons. Neurochem. Int. 22, 19\u201329 (1993).","journal-title":"Neurochem. Int."},{"key":"BFsrep33285_CR38","doi-asserted-by":"publisher","first-page":"2541","DOI":"10.1007\/s11064-012-0847-5","volume":"37","author":"C Rae","year":"2012","unstructured":"Rae, C., Fekete, A. D., Kashem, M. a., Nasrallah, F. a. & Br\u00f6er, S. Metabolism, compartmentation, transport and production of acetate in the cortical brain tissue slice. Neurochem. Res. 37, 2541\u20132553 (2012).","journal-title":"Neurochem. Res."},{"key":"BFsrep33285_CR39","doi-asserted-by":"publisher","first-page":"1260","DOI":"10.1002\/jnr.21230","volume":"85","author":"C Brito","year":"2007","unstructured":"Brito, C. et al. Increased levels of fucosyltransferase IX and carbohydrate Lewis(x) adhesion determinant in human NT2N neurons. J. Neurosci. Res. 85, 1260\u20131270 (2007).","journal-title":"J. Neurosci. Res."},{"key":"BFsrep33285_CR40","doi-asserted-by":"publisher","first-page":"147","DOI":"10.1002\/glia.21265","volume":"60","author":"E Brekke","year":"2012","unstructured":"Brekke, E. et al. Direct measurement of backflux between oxaloacetate and fumarate following pyruvate carboxylation. Glia 60, 147\u2013158 (2012).","journal-title":"Glia"},{"key":"BFsrep33285_CR41","doi-asserted-by":"publisher","first-page":"724","DOI":"10.1038\/jcbfm.2014.8","volume":"34","author":"EMF Brekke","year":"2014","unstructured":"Brekke, E. M. F. et al. The pentose phosphate pathway and pyruvate carboxylation after neonatal hypoxic-ischemic brain injury. J. Cereb. blood flow Metab. 34, 724\u2013734 (2014).","journal-title":"J. Cereb. blood flow Metab."},{"key":"BFsrep33285_CR42","doi-asserted-by":"publisher","first-page":"39","DOI":"10.1016\/j.ymben.2009.08.008","volume":"12","author":"N Carinhas","year":"2010","unstructured":"Carinhas, N. et al. Improving baculovirus production at high cell density through manipulation of energy metabolism. Metab. Eng. 12, 39\u201352 (2010).","journal-title":"Metab. Eng."},{"key":"BFsrep33285_CR43","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1186\/1472-6750-9-82","volume":"9","author":"M Serra","year":"2009","unstructured":"Serra, M., Brito, C., Costa, E. M., Sousa, M. F. Q. & Alves, P. M. Integrating human stem cell expansion and neuronal differentiation in bioreactors. BMC Biotechnol. 9, 1\u201314 (2009).","journal-title":"BMC Biotechnol."},{"key":"BFsrep33285_CR44","doi-asserted-by":"publisher","first-page":"599","DOI":"10.1038\/nmeth.2508","volume":"10","author":"EJ Gualda","year":"2013","unstructured":"Gualda, E. J. et al. OpenSpinMicroscopy: an open-source integrated microscopy platform. Nat Methods 10, 599\u2013600 (2013).","journal-title":"Nat Methods"},{"key":"BFsrep33285_CR45","doi-asserted-by":"publisher","first-page":"1","DOI":"10.3389\/fncel.2014.00221","volume":"8","author":"EJ Gualda","year":"2014","unstructured":"Gualda, E. J., Sim\u00e3o, D., Pinto, C., Alves, P. M. & Brito, C. Imaging of human differentiated 3D neural aggregates using light sheet fluorescence microscopy. Front. Cell. Neurosci. 8, 1\u201310 (2014).","journal-title":"Front. Cell. Neurosci."},{"key":"BFsrep33285_CR46","doi-asserted-by":"publisher","first-page":"676","DOI":"10.1038\/nmeth.2019","volume":"9","author":"J Schindelin","year":"2012","unstructured":"Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676\u2013682 (2012).","journal-title":"Nat. Methods"},{"key":"BFsrep33285_CR47","doi-asserted-by":"publisher","first-page":"402","DOI":"10.1006\/meth.2001.1262","volume":"25","author":"KJ Livak","year":"2001","unstructured":"Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402\u2013408 (2001).","journal-title":"Methods"},{"key":"BFsrep33285_CR48","doi-asserted-by":"publisher","first-page":"2773","DOI":"10.1046\/j.1471-4159.1995.64062773.x","volume":"64","author":"B Hassel","year":"1995","unstructured":"Hassel, B., Sonnewald, U. & Fonnum, F. Glial-neuronal interactions as studied by cerebral metabolism of [2-13C]acetate and [1-13C]glucose: an ex vivo 13C NMR spectroscopic study. J. Neurochem. 64, 2773\u20132782 (1995).","journal-title":"J. Neurochem."},{"key":"BFsrep33285_CR49","doi-asserted-by":"publisher","first-page":"2916","DOI":"10.1038\/nprot.2006.476","volume":"1","author":"Ma Gaffield","year":"2006","unstructured":"Gaffield, M. a & Betz, W. J. Imaging synaptic vesicle exocytosis and endocytosis with FM dyes. Nat. Protoc. 1, 2916\u20132921 (2006).","journal-title":"Nat. Protoc."},{"key":"BFsrep33285_CR50","first-page":"77","volume":"2012","author":"P Hoopmann","year":"2012","unstructured":"Hoopmann, P., Rizzoli, S. O. & Betz, W. J. Imaging synaptic vesicle recycling by staining and destaining vesicles with FM dyes. Cold Spring Harb. Protoc. 2012, 77\u201383 (2012).","journal-title":"Cold Spring Harb. Protoc."},{"key":"BFsrep33285_CR51","doi-asserted-by":"publisher","first-page":"e0118786","DOI":"10.1371\/journal.pone.0118786","volume":"10","author":"EK Woehrling","year":"2015","unstructured":"Woehrling, E. K. et al. A Predictive In Vitro Model of the Impact of Drugs with Anticholinergic Properties on Human Neuronal and Astrocytic Systems. PLoS One 10, e0118786 (2015).","journal-title":"PLoS One"},{"key":"BFsrep33285_CR52","doi-asserted-by":"publisher","first-page":"103","DOI":"10.1007\/s12640-009-9084-3","volume":"17","author":"EK Woehrling","year":"2010","unstructured":"Woehrling, E. K., Hill, E. J. & Coleman, M. D. Evaluation of the importance of astrocytes when screening for acute toxicity in neuronal cell systems. Neurotox. Res. 17, 103\u2013113 (2010).","journal-title":"Neurotox. Res."},{"key":"BFsrep33285_CR53","doi-asserted-by":"publisher","first-page":"2329","DOI":"10.1038\/nprot.2014.158","volume":"9","author":"MA Lancaster","year":"2014","unstructured":"Lancaster, M. A. & Knoblich, J. A. Generation of cerebral organoids from human pluripotent stem cells. Nat. Protoc. 9, 2329\u20132340 (2014).","journal-title":"Nat. Protoc."},{"key":"BFsrep33285_CR54","doi-asserted-by":"publisher","first-page":"816","DOI":"10.1126\/science.aaf6116","volume":"352","author":"PP Garcez","year":"2016","unstructured":"Garcez, P. P. et al. Zika virus impairs growth in human neurospheres and brain organoids. Science (80-.). 352, 816\u2013818 (2016).","journal-title":"Science (80-.)."},{"key":"BFsrep33285_CR55","doi-asserted-by":"publisher","first-page":"3386","DOI":"10.1002\/jnr.21409","volume":"85","author":"S S\u00e1 Santos","year":"2007","unstructured":"S\u00e1 Santos, S., Leite, S. B., Sonnewald, U., Carrondo, M. J. T. & Alves, P. M. Stirred vessel cultures of rat brain cells aggregates: characterization of major metabolic pathways and cell population dynamics. J. Neurosci. Res. 85, 3386\u20133397 (2007).","journal-title":"J. Neurosci. Res."},{"key":"BFsrep33285_CR56","doi-asserted-by":"publisher","first-page":"665","DOI":"10.1016\/j.neuint.2011.02.005","volume":"58","author":"S S\u00e1 Santos","year":"2011","unstructured":"S\u00e1 Santos, S., Sonnewald, U., Carrondo, M. J. T. & Alves, P. M. The role of glia in neuronal recovery following anoxia: In vitro evidence of neuronal adaptation. Neurochem. Int. 58, 665\u2013675 (2011).","journal-title":"Neurochem. Int."},{"key":"BFsrep33285_CR57","doi-asserted-by":"publisher","first-page":"735","DOI":"10.1111\/j.1471-4159.2010.06636.x","volume":"113","author":"AI Amaral","year":"2010","unstructured":"Amaral, A. I. et al. Metabolic alterations induced by ischemia in primary cultures of astrocytes: Merging 13C NMR spectroscopy and metabolic flux analysis. J. Neurochem. 113, 735\u2013748 (2010).","journal-title":"J. Neurochem."},{"key":"BFsrep33285_CR58","doi-asserted-by":"publisher","first-page":"1484","DOI":"10.1111\/j.1471-4159.1983.tb00849.x","volume":"41","author":"aC Yu","year":"1983","unstructured":"Yu, a C., Drejer, J., Hertz, L. & Schousboe, a. Pyruvate carboxylase activity in primary cultures of astrocytes and neurons. J. Neurochem. 41, 1484\u20131487 (1983).","journal-title":"J. Neurochem."},{"key":"BFsrep33285_CR59","doi-asserted-by":"publisher","first-page":"364","DOI":"10.1016\/0006-8993(85)90552-9","volume":"329","author":"RP Shank","year":"1985","unstructured":"Shank, R. P., Bennett, G. S., Freytag, S. O. & Campbell, G. L. Pyruvate carboxylase: an astrocyte-specific enzyme implicated in the replenishment of amino acid neurotransmitter pools. Brain Res. 329, 364\u2013367 (1985).","journal-title":"Brain Res."},{"key":"BFsrep33285_CR60","doi-asserted-by":"publisher","first-page":"303","DOI":"10.1016\/0006-8993(79)90071-4","volume":"161","author":"MD Norenberg","year":"1979","unstructured":"Norenberg, M. D. & Martinez-Hernandez, A. Fine structural localization of glutamine synthetase in astrocytes of rat brain. Brain Res. 161, 303\u2013310 (1979).","journal-title":"Brain Res."},{"key":"BFsrep33285_CR61","doi-asserted-by":"publisher","first-page":"1066","DOI":"10.1021\/bi00831a038","volume":"8","author":"Ra Ronzio","year":"1969","unstructured":"Ronzio, R. a., Rowe, W. B. & Meister, a. Studies on the mechanism of inhibition of glutamine synthetase by methionine sulfoximine. Biochemistry 8, 1066\u20131075 (1969).","journal-title":"Biochemistry"},{"key":"BFsrep33285_CR62","doi-asserted-by":"publisher","first-page":"477","DOI":"10.1016\/S0306-9877(98)90067-6","volume":"51","author":"CA Shaw","year":"1998","unstructured":"Shaw, C. A. & Bains, J. S. Did consumption of flour bleached by the agene process contribute to the incidence of neurological disease? Med. Hypotheses 51, 477\u2013481 (1998).","journal-title":"Med. Hypotheses"},{"key":"BFsrep33285_CR63","doi-asserted-by":"publisher","first-page":"1304","DOI":"10.1046\/j.1471-4159.1998.70031304.x","volume":"70","author":"K Kanamori","year":"1998","unstructured":"Kanamori, K., Ross, B. D. & Kondrat, R. W. Rate of glutamate synthesis from leucine in rat brain measured in vivo by 15N NMR. J. Neurochem. 70, 1304\u20131315 (1998).","journal-title":"J. Neurochem."},{"key":"BFsrep33285_CR64","doi-asserted-by":"publisher","first-page":"1712","DOI":"10.1046\/j.1471-4159.2001.00156.x","volume":"76","author":"E Lieth","year":"2001","unstructured":"Lieth, E. et al. Nitrogen shuttling between neurons and glial cells during glutamate synthesis. J. Neurochem. 76, 1712\u20131723 (2001).","journal-title":"J. Neurochem."},{"key":"BFsrep33285_CR65","doi-asserted-by":"publisher","first-page":"846S","DOI":"10.1093\/jn\/131.3.846S","volume":"131","author":"SM Hutson","year":"2001","unstructured":"Hutson, S. M., Lieth, E. & LaNoue, K. F. Function of leucine in excitatory neurotransmitter metabolism in the central nervous system. J. Nutr. 131, 846S\u2013850S (2001).","journal-title":"J. Nutr."},{"key":"BFsrep33285_CR66","doi-asserted-by":"publisher","first-page":"997","DOI":"10.1111\/jnc.12044","volume":"123","author":"J Hull","year":"2012","unstructured":"Hull, J. et al. Distribution of the branched chain aminotransferase proteins in the human brain and their role in glutamate regulation. J. Neurochem. 123, 997\u20131009 (2012).","journal-title":"J. Neurochem."},{"key":"BFsrep33285_CR67","first-page":"245","volume":"16","author":"J Jessy","year":"1988","unstructured":"Jessy, J. & Murthy, C. R. Branched chain amino acid transaminases in brain in methionine sulphoximine (MSI) toxicity. Biochem. Int. 16, 245\u2013251 (1988).","journal-title":"Biochem. Int."},{"key":"BFsrep33285_CR68","doi-asserted-by":"publisher","first-page":"1650","DOI":"10.1289\/ehp.1205432","volume":"120","author":"RM LoPachin","year":"2012","unstructured":"LoPachin, R. M. & Gavin, T. Molecular mechanism of acrylamide neurotoxicity: Lessons learned from organic chemistry. Environ. Health Perspect. 120, 1650\u20131657 (2012).","journal-title":"Environ. Health Perspect."},{"key":"BFsrep33285_CR69","doi-asserted-by":"publisher","first-page":"617","DOI":"10.1016\/j.neuro.2004.01.004","volume":"25","author":"RM LoPachin","year":"2004","unstructured":"LoPachin, R. M. The changing view of acrylamide neurotoxicity. Neurotoxicology 25, 617\u2013630 (2004).","journal-title":"Neurotoxicology"},{"key":"BFsrep33285_CR70","doi-asserted-by":"publisher","first-page":"422","DOI":"10.1016\/0041-008X(90)90146-L","volume":"105","author":"RL DeGrandchamp","year":"1990","unstructured":"DeGrandchamp, R. L., Reuhl, K. R. & Lowndes, H. E. Synaptic terminal degeneration and remodeling at the rat neuromuscular junction resulting from a single exposure to acrylamide. Toxicol. Appl. Pharmacol. 105, 422\u2013433 (1990).","journal-title":"Toxicol. Appl. Pharmacol."},{"key":"BFsrep33285_CR71","doi-asserted-by":"publisher","first-page":"117","DOI":"10.1016\/j.neuro.2014.03.010","volume":"43","author":"J Sisnaiske","year":"2014","unstructured":"Sisnaiske, J. et al. Acrylamide alters neurotransmitter induced calcium responses in murine ESC-derived and primary neurons. Neurotoxicology 43, 117\u2013126 (2014).","journal-title":"Neurotoxicology"},{"key":"BFsrep33285_CR72","doi-asserted-by":"publisher","first-page":"717","DOI":"10.1111\/j.1471-4159.1974.tb04285.x","volume":"22","author":"MS Patel","year":"1974","unstructured":"Patel, M. S. The relative significance of CO2 fixing enzymes in the metabolism of rat brain. J. Neurochem. 22, 717\u2013724 (1974).","journal-title":"J. Neurochem."},{"key":"BFsrep33285_CR73","doi-asserted-by":"publisher","first-page":"975","DOI":"10.1046\/j.1471-4159.2001.00074.x","volume":"76","author":"NR Sibson","year":"2001","unstructured":"Sibson, N. R. et al. In vivo 13C NMR measurement of neurotransmitter glutamate cycling, anaplerosis and TCA cycle flux in rat brain during [2-13C]glucose infusion. J. Neurochem. 76, 975\u2013989 (2001).","journal-title":"J. Neurochem."}],"container-title":["Scientific Reports"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.nature.com\/articles\/srep33285.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/srep33285","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/srep33285.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,1,5]],"date-time":"2023-01-05T00:19:45Z","timestamp":1672877985000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.nature.com\/articles\/srep33285"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2016,9,13]]},"references-count":73,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2016,12,16]]}},"alternative-id":["BFsrep33285"],"URL":"https:\/\/doi.org\/10.1038\/srep33285","relation":{},"ISSN":["2045-2322"],"issn-type":[{"type":"electronic","value":"2045-2322"}],"subject":[],"published":{"date-parts":[[2016,9,13]]},"assertion":[{"value":"18 December 2015","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"19 August 2016","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"13 September 2016","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"The authors declare no competing financial interests.","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"33285"}}