Journal: Pharmaceutics

Article Title: Comparison of the Effect of the Combination of Sodium Valproate and Sodium Dichloroacetate on the Expression of SLC12A2 , SLC12A5 , CDH1 , CDH2 , EZH2 , and GFAP in Primary Female Glioblastoma Cells with That of Temozolomide

doi: 10.3390/pharmaceutics17091161

Figure Lengend Snippet: ( A ) The SLC12A2 expression (ΔCT) of the GBM patient tumor control group, normalized to the GAPDH . ( B ) Relative SLC12A2 expression (Log 2 (2 −ΔΔCT )) of female GBM patient tumor groups tested. Exact p -values are given compared to the control.

Article Snippet: Real-time PCR for SLC12A2 (TaqMan Assay ID: Hs00169032_m1), SLC12A5 (TaqMan Assay ID: Hs00221168_m1), CDH1 (TaqMan Assay ID: Hs01023895_m1), CDH2 (TaqMan Assay ID: Hs00983056_m1), EZH2 (TaqMan Assay ID: Hs00544830_m1) and GFAP (TaqMan Assay ID: Hs00909233_m1) were carried out using TaqMan chemistry (Applied BiosystemsTM, Thermo Fisher Scientific, Roterdam, The Netherlands) following the manufacturer’s protocol on a 7900 Real-Time PCR System (Applied BiosystemsTM, Thermo Scientific, Carlsbad, CA, USA).

Techniques: Expressing, Control

Journal: PLOS Biology

Article Title: GABAergic regulation of striatal spiny projection neurons depends upon their activity state

doi: 10.1371/journal.pbio.3002483

Figure Lengend Snippet: (A) The RiboTag construct AAV5-DOI-hSyn-RpI22I1-3Xflag-2A-eGFP was injected into the striatum of Adora2a-cre mice at P18 or at 6 months of age. (B) The coronal slice images demonstrate both the coverage and restriction to the striatum of the stereotaxically injected AAV carrying the RiboTag and eGFP genes (stereotaxic injection coordinates: ML = −1.85, AP = +0.74, DV = −3.50). Scale bars = 1 mm and 20 μm. Ten days later, the infected tissue (green fluorescence) was dissected out with the aid of fluorescence microscopy and qPCR was performed. (C) mRNA abundance (ΔCT) levels for the chloride cotransporters NKCC1 ( SLC12A1 ) and KCC2 ( SLC12A5 ) were determined by qPCR in striata from Adora2a-cre mice 4 weeks and 6 months of age. The data underlying the graphs shown in the figure can be found in dx.doi.org/10.5281/zenodo.10386854 . AAV, adeno-associated virus; qPCR, quantitative polymerase chain reaction; SPN, spiny projection neuron.

Article Snippet: TaqMan probes were used for PCR amplification of Hprt , Mm03024075_m1, Slc12a2 (NKCC1), Mm01265955_m1, Slc12a5 (KCC2), Mm00803929_m1, Slc4a3 (AE3) Mm00436654_g1, and Slc4a10 (NCBE) Mm00473827_m1.

Techniques: Construct, Injection, Infection, Fluorescence, Microscopy, Virus, Real-time Polymerase Chain Reaction

Journal: PLOS Biology

Article Title: GABAergic regulation of striatal spiny projection neurons depends upon their activity state

doi: 10.1371/journal.pbio.3002483

Figure Lengend Snippet: (A) Clomeleon-expressing iSPNs allowed visual identification of dendrites in gramicidin perforated-patch recording conditions where cells cannot be loaded with dyes via internal-pipette solution (920 nm laser maximum projection image, scale bar = 40 μm). When low (<100 MΩ) access-resistance was achieved in voltage-clamp mode, RuBi-GABA (10 μM) was uncaged with a 473 nm laser spot (approximately 1 μm diameter, 1 ms) in the presence of the synaptic blockers: TTX (1 μM), AP5 (50 μM), NBQX (5 μM), CGP-55845 (1 μM). The laser was targeted to the somatic region or to distal dendrites (blue spots, projection image). (B) Representative voltage traces showing GABA responses, recorded in serial, from the soma (top traces, scale bars = 20 pA/2 s) or the dendrite (lower traces, scale bars = 10 pA/2 s) as the membrane was manually stepped from −70 mV to −50 mV. (C) Plot of the current/voltage relationship between somatic and dendritic activation. The data, represented by medians with interquartile ranges, did not differ significantly between the soma and dendritic compartments ( n = 5 each; soma, dendrite; slope = 0.96, 0.71; x-intercept = −55.9, −59.6 mV; R 2 = 0.77, 0.85, respectively). Current measurements were rounded to the nearest 0.5 pA. (D) Current-clamp experiments in gramicidin perforated-patch mode were performed to examine age-dependent shifts in reversal potential. Here, Adora2a-eGFP positive iSPNs could be visually identified and patched. When low (<100 MΩ) access-resistance was achieved in current-clamp mode, the resting membrane potential along with series of hyperpolarizing and depolarizing steps were used to examine cell health (traces, scale bars = 20 mV/200 ms). (E) RuBi-GABA (10 μM) was uncaged over the full-field (3 ms duration, 60× lens) with a 473 nm LED in the presence of the synaptic blockers: AP5 (50 μM), NBQX (5 μM), CGP-55845 (1 μM). Representative current traces showing GABA responses as the membrane was manually stepped from −80 mV to −50 mV, scale bars = 5 mV/200 ms. (F) Plot of the change in PSP amplitude at P30, P90, and P270. The data, represented by medians with interquartile ranges, did not differ significantly between the 3 ages tested ( n = 5 each P30, P90, P270; slope = 0.75, 0.70, 0.75; x-intercept = −61.6, −61.5, −61.5 mV; R 2 = 0.93, 0.91, 0.94, respectively). Values were calculated to the nearest 0.5 mV for the ΔV/V measurements. The data shows that the reversal for GABA-induced current is a full 20 mV+ above the resting membrane potential for SPNs, typically, −80 to −85 mV. (G) The addition of bumetanide (NKCC1 blocker, 10 μM) did not change the GABA A R reversal potential significantly ( n = 5, p = 0.4076). (H) Perforated patch recordings were obtained from Adora2a-eGFP iSPNs and then the reversal potential of GABA A Rs determined before and after inhibition of CA with acetazolamide (aceta, 10 μM). (I) Representative traces recorded from a visually identified iSPN from an Adora2a-eGFP mouse in gramicidin perforated patch in current-clamp mode in the synaptic blockers: AP5 (50 μM), NBQX (5 μM), CGP-55845 (1 μM), MPEP (1 μM), and CPCCOEt (50 μM). RuBi-GABA (15 μM) was uncaged using a single LED pulse (470 nm, 25 ms). The pulse was applied at an interval of 30 s while manually stepping the cell to different potentials from −80 to −50 mV, scale bars = 10 mV/100 ms. (J) Summary data shows that application of acetazolamide shifted the reversal of the GABA-induced current to more negative potentials ( p = 0.03125, n = 6). Membrane potentials were adjusted to correct for the estimated liquid junction potential and then binned into 5 mV increments (-70, -65, -60, -55 and -50 mV). The data underlying the graphs shown in the figure can be found in dx.doi.org/10.5281/zenodo.10386854 . CA, carbonic anhydrase; PSP, postsynaptic potential; SPN, spiny projection neuron.

Article Snippet: TaqMan probes were used for PCR amplification of Hprt , Mm03024075_m1, Slc12a2 (NKCC1), Mm01265955_m1, Slc12a5 (KCC2), Mm00803929_m1, Slc4a3 (AE3) Mm00436654_g1, and Slc4a10 (NCBE) Mm00473827_m1.

Techniques: Expressing, Transferring, Membrane, Activation Assay, Inhibition

Journal: bioRxiv

Article Title: NKCC1 modulates microglial phenotype, cerebral inflammatory responses and brain injury in a cell-autonomous manner

doi: 10.1101/2021.01.21.427597

Figure Lengend Snippet: A : Mice were subjected to either intraperitoneal (ip.) or intracortical (cor.) LPS injections, while NKCC1 was blocked by ip. bumetanide (Bum) administration. Central LPS injection triggers high cytokine (G-CSF, IL-1α, IL-1β) and chemokine (KC) responses in the brain compared to ip. LPS injection, which is blocked by ip. Bum administration. B : Central NKCC1 inhibition by intracortical Bum administration significantly increases GCSF and IL-1β levels. See also Supplementary Figure 1 for effects of systemic vs. central blockade of NKCC1 on LPS-induced cytokine responses in the periphery. C : Flow cytometric dot plots show that cortical administration of Bum does not affect the number of microglia (CD45 int /P5 gate), and recruitment of leukocytes (CD45 high /P4 gate), including monocytes (CD11b + , Ly6C high /P9 gate), and granulocytes (CD11b + , Ly6G high /P7 gate) upon central LPS injection. D : The main source of IL-1α and IL-1β in the brain are microglia cells. Confocal images of Cx3CR1 +/GFP brain slices show IL-1α-CD45-P2Y12R (above, red arrowheads) and IL-1β-CD45-P2Y12R (below, red arrowheads) labelled cells after cortical LPS injection-induced inflammation. All data are expressed as mean±SEM. E : NKCC1 (encoded by Slc12a2 ) and P2Y12R gene expression is downregulated in microglia isolated from adult mice 24 hours after cisterna magna LPS application. A : One-way ANOVA followed by Tukey’s multiple comparison test; * p <0.05; N=6/group; B : Unpaired t-test; * p <0.05; N=9/group; Data were pooled from two independent studies. C : One-way ANOVA followed by Tukey’s multiple comparison test; * p <0.05; N=4/group. D : Scale: 25 μm; E : Unpaired t-test; ** p <0.01, *** p <0.001; N (WT)=6, N (KO)=5; Abbreviations: veh.: vehicle; ip: intraperitoneal; cor.: cortical; Bum: bumetanide; ns: not significant

Article Snippet: The diluted anti-NKCC1 primary antibodies (NKCC1 Rb: 1:4000, #13884-1-AP, Proteintech, NKCC1 M: 1:2000, diluted in MOM-diluent; DSHB) were pre-incubated for 48 hours with brain slices from NKCC1 -/- mice, in order to remove the fraction of immunoglobulins that could potentially cause aspecific-binding.

Techniques: Injection, Inhibition, Expressing, Isolation

Journal: bioRxiv

Article Title: NKCC1 modulates microglial phenotype, cerebral inflammatory responses and brain injury in a cell-autonomous manner

doi: 10.1101/2021.01.21.427597

Figure Lengend Snippet: A: NKCC1 mRNA expression levels in newborn and adult microglia isolated from C57BL/6J mice compared to neural progenitors derived from E17 embryonic hippocampi. Note, that NKCC1 mRNA levels decrease dramatically during in vitro maintenance (DIV10). B: We generated a novel microglia-specific conditional NKCC1 KO transgenic mouse line by crossing NKCC1 fl/fl (exon 8 of the Slc12a2 gene was flanked with lox P sites) and Cx3CR1-Cre ERT2 mice. C: NKCC1 mRNA levels in isolated NKCC1 KO microglia was markedly reduced in comparison to wild-type cells. D-E: NKCC1 protein expression in a large number of randomly sampled NKCC1 KO microglia cells is markedly reduced compared to WT cells. Inserts show plasma membrane localization of NKCC1. F-G: Automated morphological analysis and maximum intensity projections of confocal images. Inserts show cells marked with white asterisks in 3D. Arrowheads indicate altered branch structure of NKCC1 KO microglia. Automated morphological analysis shows that features of NKCC1 deficient microglia significantly differ from WT microglia. A: One-way ANOVA, followed by Holm-Sidak’s post hoc test. N=3/group. **: p <0.01; n.s.: not significant C: Mann-Whitney test, N=3/group. **: p <0.01 D: Mann-Whitney test, N (WT)=142 cells from 2 mice, N (KO)=83 cells from 1 mouse. ****: p <0.0001 E: Scale: 2 μm F-G: Scale: 10 μm; Mann-Whitney test, N (WT)=78 cells from 3 mice, N (KO)=136 cells from 5 mice. **: p <0.01, ***: p <0.001, ****: p <0.0001 Abbreviations: DIV: days in vitro; n.s.: not significant; TMX: tamoxifen

Article Snippet: The diluted anti-NKCC1 primary antibodies (NKCC1 Rb: 1:4000, #13884-1-AP, Proteintech, NKCC1 M: 1:2000, diluted in MOM-diluent; DSHB) were pre-incubated for 48 hours with brain slices from NKCC1 -/- mice, in order to remove the fraction of immunoglobulins that could potentially cause aspecific-binding.

Techniques: Expressing, Isolation, Derivative Assay, In Vitro, Generated, Transgenic Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: NKCC1 modulates microglial phenotype, cerebral inflammatory responses and brain injury in a cell-autonomous manner

doi: 10.1101/2021.01.21.427597

Figure Lengend Snippet: A : Baseline NLRP3 and IL-1β mRNA expression is increased in isolated NKCC1 KO microglia compared to WT cells. B: Experimental outline of automated morphological analysis, cytokine array and flow cytometry. C-D : Automated morphological analysis show that activated NKCC1 deficient microglia are slightly smaller than their wild-type counterparts. E : LPS-induced cytokine levels are significantly higher in the cortices of microglial NKCC1 KO mice than in WT. F: Flow cytometric dot plots show that microglial NKCC1 deficiency does not alter the number of CD11b + , CD45 int microglia (P4 gate) or numbers of infiltrating CD11b + , CD45 high leukocytes (P5 gate), CD11b + , Ly6C high monocytes (P9 gate) and CD11b + , Ly6G high granulocytes (P7 gate) in response to intracortical LPS administration. See corresponding data on peripheral cytokine levels and immune cell populations in Supplementary Figure 3 . G: Increased NLRP3 and IL-1β mRNA levels are sustained in NKCC1 KO and WT microglia 24 hours after intracisternal LPS administration . A: Unpaired t-test; * p <0.05, ** p <0.01; N (WT)=6, N (KO)=5 D: Mann-Whitney test, N (WT)=171 cells from 6 mice, N (KO)= 85 cells from 4 mice, ** p <0.01, *** p <0.001 E: Mann-Whitney test, *: p <0.05; N (WT)=7, N (KO)=6 F: unpaired t-test, N (WT)=4, N (KO)=4 G: Unpaired t-test; * p <0.05; N (WT)=5, N (KO)=5; n. s.: not significant.

Article Snippet: The diluted anti-NKCC1 primary antibodies (NKCC1 Rb: 1:4000, #13884-1-AP, Proteintech, NKCC1 M: 1:2000, diluted in MOM-diluent; DSHB) were pre-incubated for 48 hours with brain slices from NKCC1 -/- mice, in order to remove the fraction of immunoglobulins that could potentially cause aspecific-binding.

Techniques: Expressing, Isolation, Flow Cytometry, MANN-WHITNEY

Journal: bioRxiv

Article Title: NKCC1 modulates microglial phenotype, cerebral inflammatory responses and brain injury in a cell-autonomous manner

doi: 10.1101/2021.01.21.427597

Figure Lengend Snippet: A : Schematic representation of experiment. Perforated patch-clamp recordings were performed on microglial cells in acute hippocampal slice preparations. Current responses to a train of voltage steps from -100 to 100 mV with 20 mV increments and a duration of 100 ms were measured in voltage-clamp mode (holding potential: -40 mV) both in normotonic, and after 5 minute perfusion with hypotonic ACSF (50% dilution). B: Example traces of recordings from WT (black: normotonic, grey: hypotonic ACSF) and NKCC1 KO (purple: normotonic, violet: hypotonic ACSF) animal. Traces show responses for -100 and +100 mV stimulations in both conditions. C: Average I-V curve responses from WT (black squares with SEM) and NKCC1 KO cells (violet circles with SEM) in normotonic (left) and after 5 minute perfusion of hypotonic ACSF (right) D: Resting membrane potential in normotonic condition of WT vs. NKCC1 KO microglial cells. E: I-V curves calculated by the subtraction of measured values in normotonic conditions from ones in hypotonic medium, resulting in I-V curves representing the currents evoked by cell-swelling due to osmotic change. F: Reversal potentials of the swelling-induced currents measured from WT (grey) or NKCC1 KO (violet) animals (left), with corresponding intracellular Cl - concentrations (right) calculated via the Nernst equation. All parametric data are expressed as mean±SEM. C: Unpaired t-test; N(WT)=8 cells from 7 animal, N(KO)=8 cell from 6 animal; *: p<0.05, **: p<0.01 D: Unpaired t-test; N(WT)=13 microglial cells from 12 mice, N(KO)=12 microglial cells from 11 mice; **: p <0.01 E: Unpaired t-test; *: p <0.05 F: Unpaired t-test; N(WT)=8 cell, N (KO)=8 cell; *: p<0.05.

Article Snippet: The diluted anti-NKCC1 primary antibodies (NKCC1 Rb: 1:4000, #13884-1-AP, Proteintech, NKCC1 M: 1:2000, diluted in MOM-diluent; DSHB) were pre-incubated for 48 hours with brain slices from NKCC1 -/- mice, in order to remove the fraction of immunoglobulins that could potentially cause aspecific-binding.

Techniques: Patch Clamp

Journal: bioRxiv

Article Title: NKCC1 modulates microglial phenotype, cerebral inflammatory responses and brain injury in a cell-autonomous manner

doi: 10.1101/2021.01.21.427597

Figure Lengend Snippet: A-B : Microglial NKCC1-deficient mice (KO) show larger infarct volume as assessed on cresyl violet-stained brain sections and more severe neurological outcome compared to wild type mice. C: Cytokine levels in the cortex do not differ 8 hours after MCAo in KO mice compared to WT. D-E: Microglial NKCC1 deletion results in higher levels of IL-1α and IL-1β 24 hours after MCAo. B: Unpaired t-test, N (WT)=7, N (KO)=9; *: p <0.05, **: p <0.01 C: Kruskall-Wallis test followed by Dunn’s multiple comparison test; N=6/group. *: p <0.05, **: p <0.01 D: Scale: 50 μm E: Mann-Whitney test, *: p <0.05; N (WT)=7, N (KO)=9. n.s.: not significant

Article Snippet: The diluted anti-NKCC1 primary antibodies (NKCC1 Rb: 1:4000, #13884-1-AP, Proteintech, NKCC1 M: 1:2000, diluted in MOM-diluent; DSHB) were pre-incubated for 48 hours with brain slices from NKCC1 -/- mice, in order to remove the fraction of immunoglobulins that could potentially cause aspecific-binding.

Techniques: Staining, MANN-WHITNEY