Journal: Pharmacology Research & Perspectives

Article Title: Combined Inhibition of TRPM 4/ NMDA Receptor Complex and Extrasynaptic NMDA Receptors Is Candidate Therapeutic Target for Suppression of Epileptic Seizures and Improvement of Cognitive Impairments

doi: 10.1002/prp2.70256

Figure Lengend Snippet: Expression of GluN2A (A) and GluN2B (B) and basal extracellular levels of L‐glutamate (C) and D‐serine (D) in 4‐weeks and 8‐weeks of age S286L‐TG and wild‐type littermate. Ordinates indicate mean ± SD ( n = 6) of (A) expression levels of GluN2A relative to GAPDH in the plasma membrane fraction (B) expression levels of GluN2B relative to GAPDH in the plasma membrane fraction, (C) basal extracellular L‐glutamate level (μM) and (D) basal extracellular D‐serine level (μM) in the frontal cortex of wild‐type (gray column) and S286L‐TG (blue column). The lower‐side panels in A and B indicate pseudo‐gel images of capillary immunoblotting. Circles indicate the values of each individual rat. * p < 0.05, relative to 4‐weeks of age (4 W) and # p < 0.05 relative to wild‐type using two‐way ANOVA with Scheffe's post hoc test. F ‐values were in (A) expression of GluN2A ( F age [1, 20] = 46.7 [ p < 0.05], F genotype [1, 20] = 5.34 [ p < 0.05], F age*genotype [1, 20] = 1.1 [ p > 0.05]), (B) expression of GluN2B ( F age [1, 20] = 22.4 [ p < 0.05], F genotype [1, 20] = 8.3 [ p < 0.05], F age*genotype [1, 20] = 2.0 [ p > 0.05]), (C) L‐glutamate level ( F age [1, 20] = 3.2 [ p > 0.05], F genotype [1, 20] = 21.2 [ p < 0.05], F age*genotype [1, 20] = 1.9 [ p > 0.05]) and (D) D‐serine level ( F age [1, 20] = 8.4 [ p < 0.05], F genotype [1, 20] = 21.6 [ p < 0.05], F age*genotype [1, 20] = 2.8 [ p > 0.05]).

Article Snippet: Primary antibodies against GAPDH (NB300‐327, RRID:AB_10001915, 1:300; Novus Biologicals, Littleton, CO, USA), GluN2A (PPS012, RRID:AB_2112297, 1:100, R&D Systems, Minneapolis, MN, USA), GluN2B (PPS013, RRID:AB_562667, 1:100, R&D Systems), cAMP response element binding protein (CREB) (#4820, 1:50, Cell Signaling Technology, Danvers, MA, USA), and pCREB (#9198, 1:50, Cell Signaling) were used.

Techniques: Expressing, Clinical Proteomics, Membrane, Western Blot

Journal: Pharmacology Research & Perspectives

Article Title: Combined Inhibition of TRPM 4/ NMDA Receptor Complex and Extrasynaptic NMDA Receptors Is Candidate Therapeutic Target for Suppression of Epileptic Seizures and Improvement of Cognitive Impairments

doi: 10.1002/prp2.70256

Figure Lengend Snippet: Effects of chronic administration of probenecid, MK‐801, memantine, and FP802 on expression of GluN2A and GluN2B in S286L‐TG and wild‐type littermates. All rats were chronically administered by vehicle (control), probenecid (PBN: 100 mg/kg/day), MK‐801 (0.1 mg/kg/day), memantine (MEM: 10 mg/kg/day) and FP802 (40 mg/kg/day) for 2‐weeks (from 6‐weeks to 8‐weeks of age). Ordinates indicate mean ± SD ( n = 6) of expression levels of GluN2A (A1‐A4) and GluN2B (B1‐B4) relative to GAPDH in wild‐type (A1‐A2, B1‐B2) and S286L‐TG (A3‐A4, B3‐B4). The right‐side panels indicate pseudo‐gel images of capillary immunoblotting. Circles indicate the values of each individual rat. * p < 0.05, relative to control using one‐way ANOVA with Scheffe's post hoc test. F ‐values regarding effects of probenecid and MK‐801 on GluN2A expression in wild‐type (A1) ( F [2, 15] = 7.8 [ p < 0.05]), GluN2A in S286L (A3) ( F [2, 15] = 19.4 [ p < 0.05]), GluN2B in wild‐type (B1) ( F [2, 15] = 12.1 [ p < 0.05]) and GluN2B in S286L‐TG (B3) ( F [2, 15] = 18.2 [ p < 0.05]). F ‐values regarding effects of memantine and FP802 on GluN2A in wild‐type (A2) ( F [2, 15] = 0.4 [ p > 0.05]), GluN2A in S286L‐TG (A4) ( F [2, 15] = 7.1 [ p < 0.05]), GluN2B in wild‐type (B2) ( F [2, 15] = 0.2 [ p > 0.05]) and GluN2B in S286L‐TG (B4) ( F [2, 15] = 4.8 [ p < 0.05]).

Article Snippet: Primary antibodies against GAPDH (NB300‐327, RRID:AB_10001915, 1:300; Novus Biologicals, Littleton, CO, USA), GluN2A (PPS012, RRID:AB_2112297, 1:100, R&D Systems, Minneapolis, MN, USA), GluN2B (PPS013, RRID:AB_562667, 1:100, R&D Systems), cAMP response element binding protein (CREB) (#4820, 1:50, Cell Signaling Technology, Danvers, MA, USA), and pCREB (#9198, 1:50, Cell Signaling) were used.

Techniques: Expressing, Control, Western Blot

Journal: Pharmacology Research & Perspectives

Article Title: Combined Inhibition of TRPM 4/ NMDA Receptor Complex and Extrasynaptic NMDA Receptors Is Candidate Therapeutic Target for Suppression of Epileptic Seizures and Improvement of Cognitive Impairments

doi: 10.1002/prp2.70256

Figure Lengend Snippet: Effects of chronic combined administration of memantine with FP802 on ADSHE seizure frequency (A), sucrose preference (B), expression of GluN2A (C1) and GluN2B (C2), and basal extracellular levels of L‐glutamate (D) and D‐serine (E) in S286L‐TG and wild‐type littermate. All rats were chronically administered by vehicle (control) and combined of memantine (MEM: 10 mg/kg/day) with FP802 (40 mg/kg/day) for 2‐weeks (from 6‐weeks to 8‐weeks of age). Ordinates indicate mean ± SD ( n = 6) of (A) ADSHE seizure frequency (count h −1 ), (B) consumption of sucrose preference (%), (C1) expression levels of GluN2A relative to GAPDH, (C2) expression levels of GluN2B relative to GAPDH, (D) basal extracellular L‐glutamate level (μM) and (E) basal extracellular D‐serine level (μM). The right‐side panels in C1‐C2 indicate pseudo‐gel images of capillary immunoblotting. Circles indicate the values of each individual rat. * p < 0.05, relative to control and # p < 0.05 relative to wild‐type using student T ‐test or one‐way or two‐way ANOVA with Scheffe's post hoc test. F ‐values were in (B) sucrose preference: MEM ( F memantine+FP802 [1, 20] = 21.3 [ p < 0.05], F genotype [1, 20] = 5.1 [ p < 0.05], F rmemantine+FP802*genotype [1, 20] = 5.3 [ p < 0.05]), (D) L‐glutamate level: ( F memantine+FP802 [1, 20] = 5.3 [ p < 0.05], F genotype [1, 20] = 15.9 [ p < 0.05], F rmemantine+FP802*genotype [1, 20] = 4.8 [ p < 0.05]), (E) D‐serine level: ( F memantine+FP802 [1, 20] = 7.6 [ p < 0.05], F genotype [1, 20] = 22.4 [ p < 0.05], F rmemantine+FP802*genotype [1, 20] = 10.4 [ p < 0.05]).

Article Snippet: Primary antibodies against GAPDH (NB300‐327, RRID:AB_10001915, 1:300; Novus Biologicals, Littleton, CO, USA), GluN2A (PPS012, RRID:AB_2112297, 1:100, R&D Systems, Minneapolis, MN, USA), GluN2B (PPS013, RRID:AB_562667, 1:100, R&D Systems), cAMP response element binding protein (CREB) (#4820, 1:50, Cell Signaling Technology, Danvers, MA, USA), and pCREB (#9198, 1:50, Cell Signaling) were used.

Techniques: Expressing, Control, Western Blot

Journal: Experimental & Molecular Medicine

Article Title: HMGCS2-dependent β-OHB/H3K9bhb ameliorates synaptic plasticity and cognition in Alzheimer’s disease

doi: 10.1038/s12276-026-01664-9

Figure Lengend Snippet: a , b , The representative immunoblots ( a ) and quantitative analyses ( b ) of Glun1, Glun2A, Glun2B and Syn1 in the hippocampus of the WT, 3xTg-AD and 3xTg-AD+β-OHB mice, n = 4 per group. c , RT–qPCR assays mRNA expression of the Glun1, Glun2A, Glun2B and Syn1 in the hippocampus of the WT, 3xTg-AD and 3xTg-AD+β-OHB mice, n = 5 per group. d , ChIP–qPCR analysis of the enrichment of H3K9bhb at Glun1, Glun2A, Glun2B, Glun2C and Syn1 promoters in the hippocampus of the WT, 3xTg-AD and 3xTg-AD+β-OHB mice, n = 5 per group. e , f , Supplementing with β-OHB could increase the density of 3xTg-AD dendritic spines detected by Golgi-cox staining; the representative images ( e ) and quantitative analysis ( f ) of spine, n = 5 per group, three fields per mice. Scale bar, 5 μm. g – j , The Sholl analysis showed the synaptic complexity of neurons after supplementing with β-OHB in 3xTg-AD mice; the representative images ( g and i ) and the quantitative analysis ( h and j ), n = 5 per group, two fields per mice. Scale bar, 50 μm. Data are shown as mean ± s.e.m. One-way ANOVA followed by Bonferroni’s post hoc test for b – d and f . Two-way ANOVA followed by Bonferroni’s post hoc test for i and k . * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, not significant.

Article Snippet: GluN2B , NMDAR2B , Poly- , 1:1000 , Proteintech , 21920-1-AP.

Techniques: Western Blot, Quantitative RT-PCR, Expressing, ChIP-qPCR, Staining

Journal: Experimental & Molecular Medicine

Article Title: HMGCS2-dependent β-OHB/H3K9bhb ameliorates synaptic plasticity and cognition in Alzheimer’s disease

doi: 10.1038/s12276-026-01664-9

Figure Lengend Snippet: a – c , The HMGCS2 upregulation promotes the protein ( a and b ) and mRNA ( c ) expression of H3K9bhb, Glun1, Glun2A, Glun2B, Syn1 and PSD95, n = 4 or 5 per group. d , e , ChIP–qPCR analyses of the enrichment of H3K9bhb at Glun1, Glun2A, Glun2B and Syn1 promoters in the primary neurons of the WT, 3xTg-AD and 3xTg-AD + HMGCS2 mice n = 5 per group ( d ) and representative gel images from ChIP–qPCR assays ( e ). f – j , The HMGCS2 upregulation promotes the expression of Syn1 (scale bar, 25 μm) ( f ); n = 10 cells per group in MAP2 immunofluorescence ( g ) and quantitative analysis ( h ), n = 10 cells per group and SYP ( i and j ), n = 10 cells per group, scale bar, 15 μm. Data are shown as mean ± s.e.m. One-way ANOVA followed by Bonferroni’s post hoc test for b – d and j . Two-way ANOVA followed by Bonferroni’s post hoc test for h . * P < 0.05, ** P < 0.01 , *** P < 0.001, **** P < 0.0001; ns, not significant.

Article Snippet: GluN2B , NMDAR2B , Poly- , 1:1000 , Proteintech , 21920-1-AP.

Techniques: Expressing, ChIP-qPCR, Immunofluorescence

Journal: Experimental & Molecular Medicine

Article Title: HMGCS2-dependent β-OHB/H3K9bhb ameliorates synaptic plasticity and cognition in Alzheimer’s disease

doi: 10.1038/s12276-026-01664-9

Figure Lengend Snippet: a , b , A western blot analysis ( a ) of hippocampal lysates shows that HMGCS2 upregulation increases the protein levels ( b ) of H3K9bhb, Glun1, Glun2A, Glun2B, Syn1 and PSD95, n = 3 per group. c , The ChIP–qPCR analysis of H3K9bhb enrichment at the promoters of Glun2A , Glun2B , Syn1 and PSD95 in the four groups, n = 5 per group. d , The mRNA levels of Glun1, Glun2A, Glun2B, Syn1 and PSD95 in the hippocampus, as determined by RT–qPCR, n = 5 per group. e , f , Golgi staining reveals increased dendritic spine density in 3xTg-AD mice following overexpression of HMGCS2; representative images ( e ) and quantification ( f ) are shown, n = 5 per group, three fields per mice. Scale bar, 5 μm. g – j , A behavioral assessment of spatial learning and memory using the MWM, NOR and contextual fear conditioning tests: area under the curve (AUC) of escape latency during MWM training of day 1–6 ( g ), escape latency on day 7 of the MWM test ( h ), NOR discrimination index ( i ), freezing time on day 7 in the contextual fear conditioning test ( j ), n = 8 per group. Data are shown as mean ± s.e.m. One-way ANOVA followed by Bonferroni’s post hoc test for b – d , f and g – j . * P < 0.05, ** P < 0.01 , *** P < 0.001, **** P < 0.0001; ns, not significant.

Article Snippet: GluN2B , NMDAR2B , Poly- , 1:1000 , Proteintech , 21920-1-AP.

Techniques: Western Blot, ChIP-qPCR, Quantitative RT-PCR, Staining, Over Expression

Journal: Brain

Article Title: Cognitive loss after brain trauma results from sex-specific activation of synaptic pruning processes

doi: 10.1093/brain/awaf293

Figure Lengend Snippet: Controlled cortical impact-induced cognitive deficits and dendritic spine alterations are dependent on microglial serine racemase (SRR) expression in male mice. ( A ) Controlled cortical impact (CCI) induces freezing deficits in male wild-type (WT) mice at 7 dpi. Golgi staining shows reduced ( B ) mature mushroom-shaped spines and increased ( C ) degenerating thin spines in males at 7 dpi. ( D ) Deficiencies in microglial D- serine in TMEM119 creErt2 :SRR fl/fl mice after CCI injury led to improved freezing behaviour as compared to SRR fl/fl mice. ( E ) Schematic of D- serine (red dots) production in homeostatic non-injured and pathological conditions. Created in BioRender. Arizanovska, D. (2025) https://BioRender.com/vl0siss . Scale bar = 1 μM. * P < 0.05. ( A – C ) Student's t -test and ( D ) two-way ANOVA with Tukey's multiple comparison test; n = 6–10. Values represent mean ± standard error of the mean. Insets show representative mushroom-shape ( B ) and thin-shape ( C ) spines. dpi = days post-infection.

Article Snippet: Tmem119 CreERT2 :SRR fl/fl and CamKII CreERT2 :Grin2b fl/fl mice were generated by crossing SRR fl/fl and Grin2b fl/fl ( C57BL/6J- Grin2b em5Lutzy /J; Jackson Laboratory, Cat. No. 032664) mice to cell-specific cre expressing microglial Tmem119 CreERT2 (C57BL/6- Tmem119 em2(EGFP)Gfng /J; Jackson Laboratory, Cat. No. 031820) and neuronal CamKII CreERT2 [B6;129S6-Tg(Camk2a-cre/ERT2)1Aibs/J; Jackson Laboratory, Cat. No. 012362] mice, respectively.

Techniques: Expressing, Staining, Comparison, Infection

Journal: Brain

Article Title: Cognitive loss after brain trauma results from sex-specific activation of synaptic pruning processes

doi: 10.1093/brain/awaf293

Figure Lengend Snippet: Microglial D- serine alters expression of NMDAR signalling components after injury in male mice. ( A , D , G and I ) Schematic illustrations in the non-injured quadpartite hippocampal synapse and at 1 dpi of SRR fl/fl ( left ) and TMEM119 creERT2 :SRR fl/fl ( right ) male mice. ( B – E ) Western blot of whole hippocampus shows a significant increase in ( B ) GluN1, ( C ) GluN2A, and ( E ) GluN2B expression at 1 dpi in SRR fl/fl male mice that is not present in microglial knockout mice. ( D ) Schematic showing the effects of increased D- serine and NMDAR subunits between genotypes. ( F – H ) Expression of ( F ) phosphorylated GluN2B and ( H ) DAPK1 is increased at 1 dpi in SRR fl/fl male mice and not until 3 dpi in TMEM119 creERT2 :SRR fl/fl male mice. ( G ) Increased GluN2B signalling blocks pro-synaptic gene expression. ( I and J ) The ratio of phosphorylated to non-phosphorylated CREB is significantly upregulated in TMEM119 creERT2 :SRR fl/fl but not SRR fl/fl male mice after injury. ( J and K ) BDNF is significantly increased at 1 and 3 dpi in TMEM119 creERT2 :SRR fl/fl male mice, prior to upregulation in SRR fl/fl mice at 3 dpi. ( L ) The ratio of phosphorylated to non-phosphorylated ERK is significantly upregulated in SRR fl/fl but not TMEM119 creERT2 :SRR fl/fl male mice after injury. * P < 0.05, ** P < 0.01, *** P < 0.001. Two-way ANOVA with Tukey's multiple comparison test; n = 4–5. Values represent mean ± standard error of the mean. astro = astrocyte; dpi = days post-infection; micro = microglia. A , D , G and J were created in BioRender. Arizanovska, D. (2025) https://BioRender.com/l9bwv23 .

Article Snippet: Tmem119 CreERT2 :SRR fl/fl and CamKII CreERT2 :Grin2b fl/fl mice were generated by crossing SRR fl/fl and Grin2b fl/fl ( C57BL/6J- Grin2b em5Lutzy /J; Jackson Laboratory, Cat. No. 032664) mice to cell-specific cre expressing microglial Tmem119 CreERT2 (C57BL/6- Tmem119 em2(EGFP)Gfng /J; Jackson Laboratory, Cat. No. 031820) and neuronal CamKII CreERT2 [B6;129S6-Tg(Camk2a-cre/ERT2)1Aibs/J; Jackson Laboratory, Cat. No. 012362] mice, respectively.

Techniques: Expressing, Western Blot, Knock-Out, Gene Expression, Comparison, Infection

Journal: Brain

Article Title: Cognitive loss after brain trauma results from sex-specific activation of synaptic pruning processes

doi: 10.1093/brain/awaf293

Figure Lengend Snippet: Inhibition of the GluN2B NMDAR subunit rescues synaptic damage and cognitive deficits after controlled cortical impact injury in male mice. ( A ) Western blot analysis of whole hippocampal lysates shows reduced GluN2B expression in CamKII creERT2 :Grin2b fl/fl male mice. ( B ) Western blot analysis showing reduced GluN2B expression in isolated plasma membrane lysate from CamKII creERT2 :Grin2b fl/fl compared to Grin2b fl/fl male mice. ( C ) Reduced spine density in Grin2b fl/fl but not CamKII creERT2 :Grin2b fl/fl male mice, resulting from reduced ( D ) mushroom-shaped spines but not ( E ) thin-shaped spines at 7 dpi. ( F ) Contextual and ( G ) cued freezing are reduced in Grin2b fl/fl but not CamKII creERT2 :Grin2b fl/fl male mice at 7 dpi. ( H ) Ro-maleate treatment leads to a rescue of mushroom- and thin-shaped spines at 3 dpi compared to vehicle treatment in male mice. * P < 0.05, ** P < 0.01, *** P < 0.001. ( A ) Student's t -test; n = 2. ( F and G ) Student's t -test was used since baseline variances in freezing were observed; n = 10–15. ( B – E and H ) Two-way ANOVA with Tukey's multiple comparison test; n = 5–6. Values represent mean ± standard error of the mean. dpi = days post-infection.

Article Snippet: Tmem119 CreERT2 :SRR fl/fl and CamKII CreERT2 :Grin2b fl/fl mice were generated by crossing SRR fl/fl and Grin2b fl/fl ( C57BL/6J- Grin2b em5Lutzy /J; Jackson Laboratory, Cat. No. 032664) mice to cell-specific cre expressing microglial Tmem119 CreERT2 (C57BL/6- Tmem119 em2(EGFP)Gfng /J; Jackson Laboratory, Cat. No. 031820) and neuronal CamKII CreERT2 [B6;129S6-Tg(Camk2a-cre/ERT2)1Aibs/J; Jackson Laboratory, Cat. No. 012362] mice, respectively.

Techniques: Inhibition, Western Blot, Expressing, Isolation, Clinical Proteomics, Membrane, Comparison, Infection

Journal: Brain

Article Title: Cognitive loss after brain trauma results from sex-specific activation of synaptic pruning processes

doi: 10.1093/brain/awaf293

Figure Lengend Snippet: Upregulation of synaptic pruning components phosphatidylserine (PS), complement C1q and GPR56 is mediated by microglial D- serine in the hippocampus of male mice . PSVue dye (red) binds externalized PS following 3-day administration to the lateral ventricle in ( A ) sham or ( B ) controlled cortical impact (CCI)-injured male mice. Anti-vGlut1 (blue) and Anti-PSD95 (green) were used to identify the pre- and postsynaptic membranes. ( C – F ) High-magnification (×63) images show minimal labelling of externalized PS in the CA1 hippocampal stratum lucidum moleculare ( SLM ) of sham-injured SRR fl/fl or Tmem119 creERT2 :SRR fl/fl male mice, while increased PS was observed in ( G – L ) SRR fl/fl but not ( M – R ) Tmem119 creERT2 :SRR fl/fl male mice. ( S – DD ) Anti-C1q expression was similar to PS expression. ( EE – HH ) Quantification measurements of PS and C1Q in the SLM and stratum radiatum ( SR ). ( II – JJ ) Hippocampal FACS microglia show elevated numbers and MFI of GPR56 expressing cells at 3 dpi in SRR fl/fl but to a lesser extent in Tmem119 creERT2 :SRR fl/fl male mice. Scale bars = ( A ) 500 μM, ( D and H ) 5 μM, ( L ) 1 μM. * P < 0.05, ** P < 0.01, *** P < 0.001. Two-way ANOVA with Tukey's multiple comparison test. ( EE – HH ) n = 3–5 mice/group, each with three sections/areas, ( II – JJ ) n = 4. Values represent mean ± standard error of the mean. dpi = days post-infection; FACS = fluorescence-activated cell sorting.

Article Snippet: Tmem119 CreERT2 :SRR fl/fl and CamKII CreERT2 :Grin2b fl/fl mice were generated by crossing SRR fl/fl and Grin2b fl/fl ( C57BL/6J- Grin2b em5Lutzy /J; Jackson Laboratory, Cat. No. 032664) mice to cell-specific cre expressing microglial Tmem119 CreERT2 (C57BL/6- Tmem119 em2(EGFP)Gfng /J; Jackson Laboratory, Cat. No. 031820) and neuronal CamKII CreERT2 [B6;129S6-Tg(Camk2a-cre/ERT2)1Aibs/J; Jackson Laboratory, Cat. No. 012362] mice, respectively.

Techniques: Expressing, Comparison, Infection, Fluorescence, FACS

Journal: Brain

Article Title: Cognitive loss after brain trauma results from sex-specific activation of synaptic pruning processes

doi: 10.1093/brain/awaf293

Figure Lengend Snippet: Female mice show reduced microglial serine racemase (SRR) expression and no overt synaptic damage after controlled cortical impact injury. ( A ) Controlled cortical impact (CCI) does not induce freezing deficits in female wild-type (WT) mice at 7 dpi. Golgi staining shows no difference in ( B ) mature mushroom-shaped spines or ( C ) degenerating thin spines at 7 dpi. ( D ) Flow cytometry indicates increased SRR expression in male and female astrocytes at 7 dpi, but ( E ) significantly less in microglia at 7 dpi. ( F and G ) SRR staining patterns in the hippocampus of male and female CCI-injured mice that colocalize with ( H and I ) GFAP+ astrocytes and ( J and K ) IBA1+ microglia. ( L and M ) Electrophysiological long-term potentiation (LTP) recordings in male and female mice at 7 dpi is associated with ( N ) reduced phosphorylated to non-phosphorylated ratio in GluN2B in female as compared to male mice at 3 dpi. ( O ) Scale bar = 1 μM. * P < 0.05, ** P < 0.01, *** P < 0.001. ( A – C ) Student's t -test; ( D and E ) Two-way ANOVA with Tukey's multiple comparison test; ( L and M ) Repeated measures two-way ANOVA. ( A ) n = 8–11; ( B – E ) n = 5–6; ( L and M ) n = 6–7; ( N ) n = 4. Values represent mean ± standard error of the mean. dpi = days post-infection.

Article Snippet: Tmem119 CreERT2 :SRR fl/fl and CamKII CreERT2 :Grin2b fl/fl mice were generated by crossing SRR fl/fl and Grin2b fl/fl ( C57BL/6J- Grin2b em5Lutzy /J; Jackson Laboratory, Cat. No. 032664) mice to cell-specific cre expressing microglial Tmem119 CreERT2 (C57BL/6- Tmem119 em2(EGFP)Gfng /J; Jackson Laboratory, Cat. No. 031820) and neuronal CamKII CreERT2 [B6;129S6-Tg(Camk2a-cre/ERT2)1Aibs/J; Jackson Laboratory, Cat. No. 012362] mice, respectively.

Techniques: Expressing, Staining, Flow Cytometry, Comparison, Infection

Journal: Brain

Article Title: Cognitive loss after brain trauma results from sex-specific activation of synaptic pruning processes

doi: 10.1093/brain/awaf293

Figure Lengend Snippet: Single-cell RNA sequencing analysis of SRR fl/fl and Tmem119 creErt2 :SRR fl/fl hippocampi. ( A ) Numbers of sham and 3 dpi cells in SRR fl/fl and Tmem119 creErt2 :SRR fl/fl mice shows increased numbers after controlled cortical impact (CCI) injury. Volcano map of ( B ) non-dividing microglia, ( C ) dividing microglia and ( D ) astrocytes, where +log 2 fold-change represents select genes upregulated in SRR fl/fl mice (red labels represent synaptic-related genes) and −log 2 fold-change represents genes upregulated in Tmem119 creErt2 :SRR fl/fl mice. ( E ) UMAP of astrocyte and microglia subclusters show little differences between sham and CCI injured SRR fl/fl and Tmem119 creErt2 :SRR fl/fl groups. Upregulation and/or downregulation of synaptic-related pathways comparing sham to CCI injury were observed in SRR fl/fl and Tmem119 creErt2 :SRR fl/fl ( F ) microglial subclusters M0 and ( H , J and L ) astrocyte subclusters A0, A1, and A3. Selective upregulated (red labels) genes and/or downregulated (blue labels) genes were observed in SRR fl/fl and Tmem119 creErt2 :SRR fl/fl ( G ) M0 microglia and ( I , K and M ) A0, A1, and A3 astrocytes. ( N ) Summary of upregulated and downregulated genes associated with SRR fl/fl and Tmem119 creErt2 :SRR fl/fl mice from microglia or astrocyte populations, where red indicates genes associated with synaptic assembly, blue indicates synaptic disassembly, checkered blue/red indicates mixed functions, and yellow indicates synaptic-related genes with poorly defined synaptic functions. ( O ) Pathway key showing the cellular component (CC) in the outer pie and biological processes (BP) in the inner pie, where consolidated pathways are colour-coded. Numbers in each pie region represent the number of consolidated gene ontology pathways. dpi = days post-infection; NC = no change in synaptic gene expression; UMAP = Uniform Manifold Approximation and Projection.

Article Snippet: Tmem119 CreERT2 :SRR fl/fl and CamKII CreERT2 :Grin2b fl/fl mice were generated by crossing SRR fl/fl and Grin2b fl/fl ( C57BL/6J- Grin2b em5Lutzy /J; Jackson Laboratory, Cat. No. 032664) mice to cell-specific cre expressing microglial Tmem119 CreERT2 (C57BL/6- Tmem119 em2(EGFP)Gfng /J; Jackson Laboratory, Cat. No. 031820) and neuronal CamKII CreERT2 [B6;129S6-Tg(Camk2a-cre/ERT2)1Aibs/J; Jackson Laboratory, Cat. No. 012362] mice, respectively.

Techniques: Single Cell, RNA Sequencing, Infection, Gene Expression