Journal: Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology

Article Title: Glutamatergic Dysfunction of Astrocytes in Paraventricular Nucleus of Thalamus Contributes to Adult Anxiety Susceptibility in Adolescent Ethanol Exposed Mice

doi: 10.1038/s41386-025-02264-3

Figure Lengend Snippet: (a) The quantification of glutamate levels in the dorsal thalamic area including PVT. (b-d) Representative expression of GLT1 (as known as EAAT2), GLAST (as known as EAAT1), and GAPDH (b) and pooled data (c-d) of western blots showing that the GLT1 expression in the PVT of AIE mice is selectively reduced compared to that of the CON mice. (e-f) Representative figures (e) and pooled data (f) confirming the reduction of GLT1 in the conditional GLT1 knockdown mice (GLT1 cHET ). (g-h) Representative figures (g) and pooled data (h) showing the selective reduction of GLT1 in astrocytes, not in neurons of PVT. (i-k) Non-invasive magnetic resonance spectroscopy (MRS) measurement showing the increase in glutamate levels in the dorsal thalamic area including PVT of GLT1 cHET . (l-q) Representative traces (l, o) and pooled data (m-n, p-q) showing the anxiogenic profiles of GLT1 cHET in the open field test (l-n) and elevated plus maze test (o-q). Female (circles) and male (triangles). Data represented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Figure 3i , 3l , and 3o were created using BioRender.com .

Article Snippet: These membranes were incubated with anti-GLT1 antibody (1:2000, Guinea pig, Millipore Sigma Cat# AB1783, RRID:AB_90949), anti-GLAST antibody (1:2000, Rabbit, Alomone Labs Cat# AGC-021, RRID:AB_2039885), anti-GAPDH antibody (1:2000, Mouse, Sigma Aldrich Cat# MAB374, RRID:AB_2107445), and anti-xCT antibody (1:1000, Rabbit, ABclonal Cat# A2413, RRID:AB_2863004) overnight at 4 °C.

Techniques: Expressing, Knockdown, Western Blot, Spectroscopy

Journal: Frontiers in Pharmacology

Article Title: Impedance-Based Phenotypic Readout of Transporter Function: A Case for Glutamate Transporters

doi: 10.3389/fphar.2022.872335

Figure Lengend Snippet: L-glutamate induces distinct mGluR2- and EAAT1-mediated responses in a TRACT assay. (A,B,D,E) Vehicle-corrected normalized Cell Index (nCI) traces of the first 15 min (A,B) or 120 min (D,E) after stimulation of cells with vehicle (PBS) or L-glutamate (L-glu) in the absence (−dox) (A,D) or presence (+dox) (B,E) of doxycycline, shown as the mean of a representative experiment performed in duplicate. (C,F) Combined concentration-response curves of L-glu on JumpIn-EAAT1-mGluR 2 cells (±dox, red) and mock-transfected JumpIn-EAAT1 cells (mock) (±dox, grey). (G) Cellular response of 100 μM L-glu on JumpIn-EAAT1-mGluR 2 cells (±dox) pretreated for 1 h with vehicle (PBS/DMSO) or 1 µM LY341495. (H) Combined concentration-response curves of L-glu on JumpIn-EAAT1-mGluR 2 cells (±dox) pretreated for 1 h with vehicle (PBS/DMSO) (data derived from ), 10 µM UCPH-101 or 10 µM TFB-TBOA. Cellular response is expressed as the net AUC of the first 15 min (C,G,H) or 120 min (F) after L-glu stimulation. Data are normalized to the response of 1 mM (C,F,H) or 100 µM (G) L-glu on JumpIn-EAAT1-mGluR 2 (−dox) cells. Data are shown as the mean ± SEM of three or four individual experiments each performed in duplicate. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; one-way ANOVA with Tukey’s post-hoc test (G) or two-way ANOVA with Šídák’s post-hoc test compared to vehicle-treated +dox cells (H) .

Article Snippet: For transfection, a codon-optimized ORF for either human EAAT1 ( SLC1A3 , Addgene #131889), EAAT2 ( SLC1A2 , Addgene #131901), EAAT3 ( SLC1A1 , Addgene #131878), EAAT4 ( SLC1A6 , Addgene #131986) or EAAT5 ( SLC1A7 , Addgene #131998) was cloned into a Gateway-compatible pJTI R4 DEST CMV TO pA expression vector with a C-terminal Twin-Strep-tag followed by an HA-tag.

Techniques: Concentration Assay, Transfection, Derivative Assay

Journal: Frontiers in Pharmacology

Article Title: Impedance-Based Phenotypic Readout of Transporter Function: A Case for Glutamate Transporters

doi: 10.3389/fphar.2022.872335

Figure Lengend Snippet: List of (−log) potency (pEC 50 ) and inhibitory potency (pIC 50 ) values of EAAT substrates and inhibitors on various cell lines in the TRACT and phenotypic assays. Potencies were determined on non-induced (−) or doxycycline (dox)-induced (+) cells in the absence (−) or presence of an inhibitor by analyzing the net area under the curve (AUC) of the first 120 min after substrate stimulation (unless stated otherwise). pIC 50 values are written in italic. N.D. = not determined.

Article Snippet: For transfection, a codon-optimized ORF for either human EAAT1 ( SLC1A3 , Addgene #131889), EAAT2 ( SLC1A2 , Addgene #131901), EAAT3 ( SLC1A1 , Addgene #131878), EAAT4 ( SLC1A6 , Addgene #131986) or EAAT5 ( SLC1A7 , Addgene #131998) was cloned into a Gateway-compatible pJTI R4 DEST CMV TO pA expression vector with a C-terminal Twin-Strep-tag followed by an HA-tag.

Techniques: Phenotypic Assay

Journal: Frontiers in Pharmacology

Article Title: Impedance-Based Phenotypic Readout of Transporter Function: A Case for Glutamate Transporters

doi: 10.3389/fphar.2022.872335

Figure Lengend Snippet: L-glutamate induces EAAT1-specific cellular responses on JumpIn-EAAT1 cells. (A) Experimental layout of an xCELLigence growth curve, the subsequent assay course and data analysis. Shown are traces of two separate wells from a representative experiment. (B,C) Vehicle-corrected nCI traces of cells in the absence (−dox) (B) or presence (+dox) (C) of doxycycline stimulated with vehicle (PBS) or L-glutamate (L-glu), shown as the mean of a representative experiment performed in duplicate. (D) Combined concentration-response curves of L-glu on JumpIn-EAAT1 cells (±dox). (E) Combined concentration-inhibition curves of TFB-TBOA and UCPH-101 on +dox cells pretreated for 1 h with vehicle (PBS/DMSO) or increasing concentrations of TFB-TBOA or UCPH-101 and subsequently stimulated with a submaximal concentration (1 mM) of L-glu. (F) Combined concentration-response curves of L-glu on +dox cells pretreated for 1 h with vehicle (PBS/DMSO), 1 μM, 3.16 µM or 10 µM UCPH-101. (G) Combined concentration-response curves of L-glu on +dox cells pretreated for 1 h with vehicle (PBS/DMSO), 10 nM, 100 nM or 1 µM TFB-TBOA. Cellular response is expressed as the net AUC of the first 120 min after L-glu stimulation. Data are normalized to the response of 1 mM L-glu on +dox cells. Data are shown as the mean ± SEM of three to ten individual experiments each performed in duplicate.

Article Snippet: For transfection, a codon-optimized ORF for either human EAAT1 ( SLC1A3 , Addgene #131889), EAAT2 ( SLC1A2 , Addgene #131901), EAAT3 ( SLC1A1 , Addgene #131878), EAAT4 ( SLC1A6 , Addgene #131986) or EAAT5 ( SLC1A7 , Addgene #131998) was cloned into a Gateway-compatible pJTI R4 DEST CMV TO pA expression vector with a C-terminal Twin-Strep-tag followed by an HA-tag.

Techniques: Subsequent Assay, Concentration Assay, Inhibition

Journal: Frontiers in Pharmacology

Article Title: Impedance-Based Phenotypic Readout of Transporter Function: A Case for Glutamate Transporters

doi: 10.3389/fphar.2022.872335

Figure Lengend Snippet: Substrate-dependent uptake via EAAT1 mediates Na + /K + -ATPase (NKA)-dependent cell spreading. (A) Vehicle-corrected nCI traces of +dox cells stimulated with vehicle (PBS), 1 mM of L-glutamate (L-glu), D-glutamate (D-glu), L-aspartate (L-asp) or D-aspartate (D-asp), shown as the mean of a representative experiment performed in duplicate. (B) Combined concentration-response curves of L-glu (derived from ), D-glu, L-asp and D-asp on +dox cells. (C) Vehicle (PBS)-corrected nCI traces of +dox cells pretreated for 1 h with vehicle (PBS/DMSO, black) or increasing concentrations of ouabain and subsequently stimulated with 1 mM L-glu, shown as the mean of a representative experiment performed in duplicate. (D) Combined concentration-inhibition curves of ouabain in +dox cells. Vehicle (PBS)-induced responses were subtracted from L-glu-induced responses for each concentration of ouabain. Cellular response is expressed as the net AUC of the first 120 min after substrate stimulation. Data are normalized to the response of 1 mM L-glu. Data are shown as the mean ± SEM of three to six individual experiments each performed in duplicate. (E) Representative confocal images of JumpIn-EAAT1-LifeAct-GFP (green) cells 0, 30, 60, and 120 min after stimulation with vehicle (PBS) or 1 mM L-glu, scale bar = 20 µm. Stills were selected from a representative live imaging movie from two independent experiments each performed in triplicate. White arrow indicates representative behavior of a single cell, showing increased cell spreading over time.

Article Snippet: For transfection, a codon-optimized ORF for either human EAAT1 ( SLC1A3 , Addgene #131889), EAAT2 ( SLC1A2 , Addgene #131901), EAAT3 ( SLC1A1 , Addgene #131878), EAAT4 ( SLC1A6 , Addgene #131986) or EAAT5 ( SLC1A7 , Addgene #131998) was cloned into a Gateway-compatible pJTI R4 DEST CMV TO pA expression vector with a C-terminal Twin-Strep-tag followed by an HA-tag.

Techniques: Concentration Assay, Derivative Assay, Inhibition, Imaging

Journal: Frontiers in Pharmacology

Article Title: Impedance-Based Phenotypic Readout of Transporter Function: A Case for Glutamate Transporters

doi: 10.3389/fphar.2022.872335

Figure Lengend Snippet: Changes in intracellular metabolite levels upon substrate stimulation of JumpIn-EAAT1 cells. Targeted metabolomics was used to measure the concentrations of several metabolites in the absence (−dox) or presence (+dox) of doxycycline. AMP, adenosine monophosphate; cAMP, cyclic AMP; IMP, inosine monophosphate. Cells were pretreated for 1 h with vehicle (PBS/DMSO, plain bars) or 10 µM TFB-TBOA (hatched bars) prior to stimulation with vehicle (PBS, white bars), 1 mM L-glutamate (red bars) or L-aspartate (blue bars). Data are shown as the mean concentration (in µM) ± SD of four replicate experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; one-way ANOVA with Tukey’s post-hoc test.

Article Snippet: For transfection, a codon-optimized ORF for either human EAAT1 ( SLC1A3 , Addgene #131889), EAAT2 ( SLC1A2 , Addgene #131901), EAAT3 ( SLC1A1 , Addgene #131878), EAAT4 ( SLC1A6 , Addgene #131986) or EAAT5 ( SLC1A7 , Addgene #131998) was cloned into a Gateway-compatible pJTI R4 DEST CMV TO pA expression vector with a C-terminal Twin-Strep-tag followed by an HA-tag.

Techniques: Concentration Assay

Journal: Frontiers in Pharmacology

Article Title: Impedance-Based Phenotypic Readout of Transporter Function: A Case for Glutamate Transporters

doi: 10.3389/fphar.2022.872335

Figure Lengend Snippet: L-glutamate induces cellular responses on MDA-MB-468 cells with endogenous EAAT1 expression. (A,B) Vehicle-corrected nCI traces of MDA-MB-468 (A) and 1321N1 (B) cells stimulated with L-glutamate (L-glu), shown as the mean of a representative experiment performed in duplicate. (C) Combined concentration-response curve of L-glu on MDA-MB-468 cells. (D) Cellular response of L-glu or L-aspartate (L-asp) on MDA-MB-468 and 1321N1 cells. MDA-MB-468 cells were pretreated for 1 h with vehicle (PBS/DMSO), 10 µM TFB-TBOA, 10 µM UCPH-101 or 1 µM ouabain prior to stimulation with vehicle (PBS), 1 mM L-glu or L-asp. Data are shown as the mean ± SEM of three (MDA-MB-468) or SD of two (1321N1) individual experiments each performed in duplicate. *** p < 0.001; one-way ANOVA with Dunnett’s post-hoc test.

Article Snippet: For transfection, a codon-optimized ORF for either human EAAT1 ( SLC1A3 , Addgene #131889), EAAT2 ( SLC1A2 , Addgene #131901), EAAT3 ( SLC1A1 , Addgene #131878), EAAT4 ( SLC1A6 , Addgene #131986) or EAAT5 ( SLC1A7 , Addgene #131998) was cloned into a Gateway-compatible pJTI R4 DEST CMV TO pA expression vector with a C-terminal Twin-Strep-tag followed by an HA-tag.

Techniques: Expressing, Concentration Assay

Journal: Frontiers in Pharmacology

Article Title: Impedance-Based Phenotypic Readout of Transporter Function: A Case for Glutamate Transporters

doi: 10.3389/fphar.2022.872335

Figure Lengend Snippet: L-glutamate-induced cellular responses on JumpIn-EAAT2 and JumpIn-EAAT3 cells. (A – D , G , H) Vehicle-corrected nCI traces of JumpIn-EAAT2 (A , B) , JumpIn-EAAT3 (C , D) , JumpIn-EAAT4 (G) and JumpIn-EAAT5 (H) cells in the absence (−dox) or presence (+dox) of doxycycline stimulated with vehicle (PBS) or L-glutamate (L-glu), shown as the mean of a representative experiment performed in duplicate. (E) Combined concentration-response curves of L-glu on JumpIn-EAAT2 and JumpIn-EAAT3 cells (±dox). Data are normalized to the response of 1 mM L-glu on JumpIn-EAAT1 +dox cells (data is derived from and shown as a black dotted line). (F) Combined concentration-inhibition curves of TFB-TBOA on +dox JumpIn-EAAT2 and JumpIn-EAAT3 cells pretreated for 1 h with vehicle (PBS/DMSO) or increasing concentrations of TFB-TBOA and subsequently stimulated with a submaximal concentration (316 µM) of L-glu. Data are normalized to the response of 1 mM L-glu (JumpIn-EAAT1, data is derived from and shown as a black dotted line) or 316 μM L-glu (JumpIn-EAAT2 and -EAAT3). (I) Cellular response of 1 mM L-glu on−dox and +dox JumpIn cells for all EAAT subtypes. Data on JumpIn-EAAT1, -EAAT2 and -EAAT3 cells were derived from and . Cellular response is expressed as the net AUC of the first 120 min after substrate stimulation. Data are shown as the mean ± SEM of at least three individual experiments each performed in duplicate. ns = not significant, ** p < 0.01, **** p < 0.0001; unpaired two-tailed Student’s t-test.

Article Snippet: For transfection, a codon-optimized ORF for either human EAAT1 ( SLC1A3 , Addgene #131889), EAAT2 ( SLC1A2 , Addgene #131901), EAAT3 ( SLC1A1 , Addgene #131878), EAAT4 ( SLC1A6 , Addgene #131986) or EAAT5 ( SLC1A7 , Addgene #131998) was cloned into a Gateway-compatible pJTI R4 DEST CMV TO pA expression vector with a C-terminal Twin-Strep-tag followed by an HA-tag.

Techniques: Concentration Assay, Derivative Assay, Inhibition, Two Tailed Test

Journal: Frontiers in Pharmacology

Article Title: Impedance-Based Phenotypic Readout of Transporter Function: A Case for Glutamate Transporters

doi: 10.3389/fphar.2022.872335

Figure Lengend Snippet: Proposed mechanism of the phenotypic impedance-based assay. Cells (over)expressing excitatory amino acid transporters (e.g., EAAT1) are stimulated with exogenous substrate (e.g., glutamate). The substrate is taken up into the cell through EAAT together with 3 Na + in exchange for 1 K + . Cl − enters the cells via uncoupled Cl − -conductivity of EAAT or other Cl − influx mechanisms, counterbalancing the increase in intracellular Na + . The Na + /K + -ATPase (NKA) restores the transmembrane Na + gradient by ATP-dependent efflux of 3 Na + in exchange for 2 K + . Over time, the intracellular concentrations of substrate, Na + , K + , and Cl − ions initially rise, increasing cell osmolarity. Subsequent cell swelling, via influx of H 2 O into the cytosol, triggers cell spreading, which increases the surface area of the E-plate that is covered by cells. Regulatory mechanisms lead to dissipation of the high intracellular ion concentrations via channels and/or transporters that mediate efflux of Cl − , K + , and osmolytes such as taurine, effectively reducing cell volume over time.

Article Snippet: For transfection, a codon-optimized ORF for either human EAAT1 ( SLC1A3 , Addgene #131889), EAAT2 ( SLC1A2 , Addgene #131901), EAAT3 ( SLC1A1 , Addgene #131878), EAAT4 ( SLC1A6 , Addgene #131986) or EAAT5 ( SLC1A7 , Addgene #131998) was cloned into a Gateway-compatible pJTI R4 DEST CMV TO pA expression vector with a C-terminal Twin-Strep-tag followed by an HA-tag.

Techniques: Expressing