Table Info

Table 1.

Experimental aspects used in published excitotoxicity papers

Ref	Model (cell line)	Species	Medium	Inductor	Concentration	Treatment time	Time after induction	Methods for validation	Outcome
[31]	Primary cortical neurons	Rat	DMEM high glucose, Neurobasal	Glutamate	10, 50 and 100 μmol/L	20 min	24 h	Morphology analysis and ELISA	Decreased cell survival rate
[32]	Primary cortical neurons	Rat	DMEM high glucose, Neurobasal	Glutamate	50 and 100 μmol/L	24 h	24 h	TUNEL, CCK-8 and [Ca²⁺]_i measurement	Decreased cell survival rate, augmented [Ca²⁺]_i and increased number of TUNEL cells
[33]	Primary cortical neurons	Mouse	Neurobasal	NMDA	50 and 100 μmol/L	10 min	24 h	Trypan blue and LDH assay	Decreased cell survival rate, increased LDH release
[34]	Primary cortical neurons	Rat	Neurobasal	NMDA	50 μmol/L	20 min	20 h	LDH assay, Hoechst-33342 staining, patch-clamp and WB	Increased LDH release; fragmented chromatin blockade of the NMDA-induced currents and cleaved caspase-3 reactivity
[35]	Primary hippocampal neurons	Rat	Neurobasal	NMDA	100 μmol/L	24 h	24 h	LDH and MTT assays, Hoechst-33342 and PI staining, Ca²⁺ imaging and patch-clamp	Decreased MTT metabolization; increased LDH release, increased [Ca²⁺]_i, fragmented chromatin and inward current
[36]	Primary cortical neurons	Rat	Neurobasal	NMDA	15, 20, 30, 40 and 50 μmol/L	30 min	20 h	WB, LDH assay, DAPI staining and Ca²⁺ imaging	Increased [Ca²⁺]_i, higher LDH release; cleaved caspase 3 reactivity, and increased condensed chromatin staining
[37]	PC12	Rat	RPMI	Glutamate	2, 5 and 10 mmol/L	48 h	48 h	MTT assay	Reduced MTT metabolization
[38]	HT22	Mouse	DMEM high glucose	Glutamate	5 mmol/L	14 h	14 h	WST-1 assay, viable cell counting, ROS analysis and WB	Increased ROS production; higher Bax and cleaved caspase-3 expression and decreased Bcl-1 expression and decreased cell survival
[39]	Organotypic hippocampal slice	Rat	Not informed	Kainate	5 μmol/L	24 h	24 h	PI staining and WB	Increased percentage of PI⁺ cells, and higher expression of BAX and PARP proteins
[40]	Primary cortical neurons	Mouse	Neurobasal	Kainate	100, 200, 400, and 800 μmol/L	1, 2, 4 and 8 h	8 h	CCK-8, oxygen consumption, mitochondrial superoxide measurement, membrane potential and ATP production	Augmented mitochondrial superoxide production, decreased mitochondrial membrane potential, reduced ATP production and decreased survival rate
[41]	Primary cortical neurons	Rat	Neurobasal	Glutamate	50, 100, 200, 500 and 1,000 mmol/L	30 min	24 h	LDH assay, morphology analysis and viable cell counting	Decreased cell survival rate and higher LDH release
[42]	Primary cerebellar granule neurons	Rat	BASAL MEDIUM EAGLE	Glutamate	1, 10, 20, 100, 250 and 500 μmol/L	24 h	24 h	MTT assay	Decreased MTT metabolization
[43]	Primary hippocampal neurons	Rat	Not informed	Glutamate	100 μmol/L	24 h	24 h	TUNEL assay and mitochondrial activity measurement	Increased TUNEL⁺ cells and collapsed mitochondrial activity
[44]	Primary cortical neurons and co-culture with primary astrocytes	Rat	DMEM high glucose	Glutamate	10, 25, 50, 100, 250, 500, and 1,000 μmol/L	10 min	72 h	Immunostaining	Increased glutamate transporter expression and decreased MAP2⁺ neurons
[45]	Primary cortical neurons	Rat	Neurobasal	Glutamate	30 μmol/L	3 h	3 h	MTT assay, immunostaining and Ca²⁺ imaging	Diminished MAP2⁺ cells, higher Ca²⁺ influx
[46]	Mesencephalic cells	Rat	MEM and Ham’s F-12	Glutamate	150 μmol/L	24 h	24 h	Immunostaining	Diminished number of PHRF-1⁺ and TH⁺ cells
[47]	Dopaminergic differentiated SH-SY5Y	Human	HBSS	Glutamate, NMDA, kainate, AMPA and (1S, 3R)- ACPD	Glutamate: 0.1, 0.5, 1, 1.5, 2, 3 and 4 mmol/L; NMDA, kainate, AMPA and (1S, 3R)- ACPD: 0.1, 0.5, 0.75 and 1 mmol/L	96 h	96 h	MTT assay and trypan blue test	Augmented trypan blue stained cells with and decreased MTT metabolization
[48]	HT22	Mouse	DMEM	Glutamate	3 and 5 mmol/L	2 h and 18 h	2 h and 18 h	Morphology analysis, MTT assay, total ATP measurement and annexin-V/PI staining	Mitochondrial fission and fragmentation, mitochondrial depolarization; decreased ATP levels, decreased MTT metabolization and higher number of annexin⁺ cells
[49]	Primary embryonic hippocampal neurons	Rat	DMEM	Synthetic Aβ 1–42	0.1, 1, 10, 50, and 100 μg/mL	24 h	24 h	Cell viability based on morphology and photo negative projection	Decreased cell viability
[50]	HEK-293 cells-huntington model	Human	MEM	NMDA and glutamate	NMDA: 3, 10, 30, 100 and 1,000 μmol/L; Glutamate: 100 μmol/L	6 h	N/A	β-galactosidase assay, Hoechst 33258 staining	Decreased cell survival rate, increased apoptotic nuclear morphology
[51]	Primary cortical neurons	Mouse	Neurobasal	NMDA and glutamate	NMDA: 100 μmol/L and Glutamate: 100 μmol/L	350 s, 6 min and 1 h	N/A	Ca²⁺ imaging, oxygen consumption and ATP measurement	Increased [Ca²⁺]_i and ATP and oxygen consumption
[52]	Primary striatal neurons	Mouse	Neurobasal	NMDA	500 μmol/L	10 min	24 h	TUNEL assay	Increased percentage of TUNEL⁺ cells
[53]	Striatal neuronal progenitor cell lines	Mouse	DMEM	NMDA	500 and 50 μmol/L	30 min	24 h	Hoechst 33258 staining	Decreased cell survival rate
[54]	Fetal primary neurons	Human	Not informed	Glutamate	10 mmol/L	24 h	24 h	DAPI staining	Decreased cell survival rate
[55]	Primary cortical neurons	Rat	Neurobasal	Glutamate	100 μmol/L	20 min	N/A	Ca²⁺ imaging	Increased [Ca²⁺]_i and mitochondrial depolarization
[56]	Primary hippocampal and striatal neurons	Rat	Not informed	NMDA	100 μmol/L	1 h	24 h	ApoAlert MitoSensor Kit and whole-cell patch clamp	Augmented number of apoptotic cells and inhibition of the NMDA currents
[57]	Primary hippocampal, cortical and midbrain neurons and glial cells	Rat	MEM	Glutamate	5 and 100 μmol/L	2.5, 3.5, 14, 15, 16, 20, 25 and 40 min	24 h	Ca²⁺ imaging, PI and Hoechst 33258 staining	Increased [Ca²⁺]_i, decreased cell survival rate
[58]	Primary cerebellar neurons	Rat	Neurobasal	Glutamate	1, 2 and 5 mmol/L	24 h	24 h	Immunostaining and WB	Downregulated expression of neuronal and anti-apoptotic markers, enhanced expression of inflammatory and senescence markers and mitochondrial damage
[59]	Embryonic stem cell 8-week derived neurons	Human	MEM	Glutamate	200 μmol/L	24 h	24 h	Ca²⁺ imaging, whole-cell patch clamp and CellTiter-Glo Luminescent Cell Viability Assay	Higher peak Ca²⁺ current, and decreased cell survival rate
[60]	Primary striatal neurons	Mouse	Neurobasal	NMDA	500 and 3,000 μmol/L	10 and 30 min	3 and 24 h	TUNEL assay, Ca²⁺ imaging, caspase activity, whole-cell patch clamp and mitochondrial membrane potential measurement	Increased [Ca²⁺]_i, increased number of TUNEL⁺ cells, mitochondrial membrane depolarization increased NMDAR-evoked current higher activity of caspase 9 and 3
[61]	Primary co-culture of cortical or hippocampal neurons and astrocytes	Rat	Neurobasal	Glutamate	100 μmol/L	3, 6, 8, 10, 12, 16, 20, 25, and 90 min	N/A	Mitochondrial membrane potential measurement, Ca²⁺ imaging, PI and Hoechst 33258 staining	Mitochondrial depolarization, increased [Ca²⁺]_i, and decreased cell survival rate
[62]	Primary hippocampal neurons	Rat	Neurobasal	Glutamate	100 μmol/L	24 h	N/A	PI and Hoechst 33342 staining, LDH assay, MTT assay, Ca²⁺ imaging, WB	Decreased cell survival rate, mitochondrial dysfunction, increased [Ca²⁺]_i, increased expression of procaspase-3 and reduced of Bcl-2
[63]	Primary hippocampal and cortical neurons	Rat	MEM	(S)-(−)- Bay K 8644, FPL 64176, NMDA and glutamate	(S)-(−)-Bay K 8644: 1 μmol/L; FPL 64176: 5 μmol/L; NMDA: 100 μmol/L; Glutamate: 200 μmol/L	5 and 30 min	20 and 24 h	PI staining, immunostainin, whole-cell recording patch clamp, electron microscopy and Ca²⁺ imaging	Increased [Ca²⁺]_i and mitochondrial dysfunction
[64]	Primary hippocampal neurons and glial cells	Rat	Neurobasal	Glutamate	Glutamate: 100 μmol/L; glycine: 10 μmol/L	3, 4, 10, 12, 14, 16, 25, 30, 35, 40 min and 24 h	24 h	Ca²⁺ imaging, PI and Hoechst 33342 staining	Mitochondrial depolarization and decreased cell survival rate
[65]	Primary neuron- astrocyte co-culture	Mouse	Neurobasal	NMDA	100 μmol/L	25 min and 24 h	N/A	Ca²⁺ imaging, immunostaining, WB and Hoechst 33258 staining	High superoxide production, increased [Ca²⁺]_i, DNA damage and decreased cell survival rate
[66]	Primary hippocampal neurons	Mouse	Neurobasal	NMDA	20 μmol/L	10 min	18 and 20 h	Hoechst 33258 staining, Ca²⁺ imaging and whole-cell patch clamp	Decreased cell survival rate, enhanced Ca²⁺ signals and mitochondria membrane depolarization

DMEM: Dulbecco’s Modified Eagle’s medium; ELISA: enzyme linked immunosorbent assay; TUNEL: transferase dUTP nick end labelling; CCK-8: cell counting kit-8; LDH: lactate dehydrogenase; WB: western blotting; MTT: methyl thiazolyl tetrazolium; PI: propidium iodide; DAPI: 4’,6-diamidino-2-phenylindole; PC12: pheochromacytoma 12; RPMI: risk of permanent medical impairment; WST-1: water-soluble tetrazolium salt-1; BAX: B-cell lymphoma-2-associated X; Bcl-1: B-cell lymphoma 1; PARP: poly(ADP-ribose) polymerase; MEM: minimal essential medium; ACPD: aminocyclopentane-1,3-dicarboxylic acid; MAP2⁺ cells: Microtubule-associated protein 2-positive cells; PHRF-1⁺ cells: PHD and RING finger domain-containing protein 1-positive cells; TH⁺ cells: tyrosine hydroxylase-positive cells; HBSS: Hanks’ balanced salt solution

Declarations

Author contributions

TG conceived the main hypothesis of the review. TG, JBS, GAJ, PNM, NT, LLP, LBM, DER, QY contributed to the manuscript writing and figures’ design. GAJ did the computational analysis. TG and JBS contributed to the formatting. TG, YT and HU reviewed the paper.

Conflicts of interest

The authors declare that they have no conflicts of interest.

Ethical approval

Not applicable.

Consent to participate

Not applicable.

Consent to publication

Not applicable.

Availability of data and materials

Not applicable.

Funding

HU acknowledges grant support from the São Paulo Research Foundation (FAPESP, Project Nos. 2018/07366-4 and 2018/08426-0) and the National Council for Scientific and Technological Development for fellowship support (CNPq, References Project No. 306392/2017-8). DER and TG are grateful for postdoctoral fellowships granted by FAPESP (Project Nos. 2018/17504-5 and 2015/13345-1-PD, respectively). LLP is grateful for a CNPq DTI-A fellowship (CNPq 380405/2020-2). The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.

Copyright

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