dio hm4di mcherry  (Addgene inc)

Journal: Science Advances

Article Title: Hcn1-dependent engram neurons in the PVN encode gastric inflammatory sensitization

doi: 10.1126/sciadv.aeb6961

Figure Lengend Snippet: ( A ) Schematic of the calcium imaging experimental setup. ( B ) Representative calcium signal from PVN neurons in the gastritis group and the saline group. ( C and D ) Gastritis-induced increase in amplitude (Δ F / F ) and frequency of the calcium signal in PVN neurons (saline versus gastritis; n = 6 mice; two-tailed t test). ( E ) RAM (receptor-activated mapping) activity tagging system. hM4Di labels neurons activated after DOX withdrawal. ( F ) Schematic of chemogenetic inhibition targeting gastritis-activated Fos PVN neurons. rAAV-F-RAM-d2tTA-hM4Di was injected into the mouse PVN region. The mice were then maintained on DOX-containing diet for 2 weeks before modeling to label gastritis-activated Fos PVN neurons. After a 3-week recovery period, intraperitoneal (ip) injection of CNO (3 mg/kg) was administered to inhibit Fos PVN neurons before gastritis modeling. ( G and H ) Chemogenetic suppression significantly reduced Fos expression in PVN compared to controls ( n = 6 mice; two-tailed t test). ( I to K ) Gastric tissue injury scores and representative H&E-stained sections ( n = 6; two-tailed t test). ( L ) Chemogenetic inhibition of Fos PVN neurons attenuated gastritis-associated inflammatory cytokine levels versus controls ( n = 6; two-tailed t test). ( M ) Schematic of optogenetic inhibition of Fos PVN neurons. ( N to P ) Gastric injury scores and H&E staining ( n = 6; two-tailed t test). ( Q ) Optogenetic inhibition reduced inflammatory cytokines compared to the control group ( n = 6; two-tailed t test).

Article Snippet: rAAV-F-RAM-d2tTA-hM4Di (200 nl per side, GeneChem) was injected bilaterally into the PVN or PSTh.

Techniques: Imaging, Saline, Two Tailed Test, Activity Assay, Inhibition, Injection, Expressing, Staining, Control

Journal: eLife

Article Title: Subregional activity in the dentate gyrus is amplified during elevated cognitive demands

doi: 10.7554/eLife.109611

Figure Lengend Snippet: ( A ) Percentage of BrdU-labeled cells that are also Dock10+ at different time points following BrdU injections (in days). ( B ) Representative section showing BrdU+ labeling (green) and Dock10+ labeling (red) in Dock10 cre;Ai9 mice. Calibration bars: 10 μm. ( C ) Schematic representation of the pattern separation paradigm. All mice (males n = 17, females n = 27) received DCZ i.p. injections 30 min before the beginning of their LS trials. ( D ) Percentage of mice reaching criterion in (S) and percentage that were not successful (NS) after 14 days of training in S configuration of LS. A majority of mice never succeeded in reaching the criterion in the Cre+ DCZ group ( z = 2.00, p = 0.0453, z -test—Group NS). ( E ) Percentage of mice reaching the criterion in S trials across days represented as the cumulative probability including all successful and not successful mice in the group. ( F ) Density of c-Fos+ GCs in the dorsal DG across the two groups. A significantly reduced density was observed in the Cre+ DCZ group compared to controls (unpaired t -test, t (9) = 2.30, p = 0.047). ( G ) Density of c-Fos+ GCs in the dorsal CA3 across the two groups. A significant reduction was observed in the Cre+ DCZ group compared to the control groups (unpaired t -test, t (9) = 4.93, p = 0.0008). ( H ) Blade-specific distribution of c-Fos+ GCs in the IB and SB of the dorsal DG in mice performing LS separation. Two-way ANOVA (unbalanced, Type II) revealed a significant main effect of blade ( F (1,16) = 315.83, p = 5.87 × 10 − ¹²), but no significant main effect of group ( F (1,16) = 0, p = 1) and no significant group × blade interaction ( F (1,16) = 0.040, p = 0.845). ( I ) Example of dorsal DG section from Dock10-cre:hM4Di mice after LS training. c-Fos+ neurons are labeled in red. Calibration bars: 100 μm. ( J ) Spatial distribution of c-Fos+ along the hilar to outer radial granule cell layer (GCL) axis of the DG (0–120 μm), with 0 μm indicating location at the subgranular zone (SGZ). ( K ) Cartoon illustrating the distribution of activity labeled GCs in the SB and IB blades of the DG. GCs are depicted in gray, while red circles represent activated cells in the DG. The overall distribution of neurons in mice in which mGCs were inhibited (Cre+ DCZ) and which did not reach criterion was not different from controls even though the total density of activity labeled neurons was lower.

Article Snippet: Strain, strain background ( Mus musculus ) , R26-LSL-Gi-DREADD (hM4Di) , Jackson Laboratory , JAX 026219 , Chemogenetic inhibition model.

Techniques: Labeling, Control, Activity Assay

Journal: eLife

Article Title: Subregional activity in the dentate gyrus is amplified during elevated cognitive demands

doi: 10.7554/eLife.109611

Figure Lengend Snippet: ( A ) Schematic representation of the pattern separation paradigm. Prior to the onset of the training, mice (males n = 17, females n = 16) were provided tamoxifen-containing food ad libitum in the home cage for 7 weeks to activate TAM inducible Cre recombinase (Ascl1-CreER T2 ) in abDGCs. All mice received DCZ (deschloroclozapine, 50 μg kg −1 ) or DMSO injections 30 min before the beginning of LS trials. ( B ) Example sections from Ascl1-CreER T2 ; hM4Di mice with immunohistochemical localization of the HA tagged DREADD (top) and after LS training with c-Fos+ neurons labeled in red. Calibration bars: 140 μm. ( C ) Percentage of mice reaching the criterion in the S trials across days represented as the cumulative probability. The Cre+ DCZ treated group needed additional days to reach criterion compared to controls. ( D ) Number of days for each mouse to reach the 70% success criterion in S trials (one-way ANOVA, F (2,31) = 3.77, p = 0.034; Tukey post hoc test indicates that the Cre+ DCZ group is significantly different from the Cre+ DMSO, while other pairs are not significantly different). ( E ) c-Fos+ cell density in all groups of mice. There was an increase in c-Fos+ mGCs in mice in which ≤7-week abDGCs were inhibited by DCZ ( H (2) = 7.22, p = 0.0271; Kruskal–Wallis) (Cre+ DCZ comparison to Cre+ DMSO, p = 0.014; Cre+ DCZ comparison to Cre- DCZ, p = 0.027 with Dunn’s multiple comparison post hoc analysis). ( F ) Example section after c-Fos immunohistochemistry to assess the distribution of c-Fos+ mGCs between the IB and SB of the dorsal DG in groups of mice who received DCZ (cre− vs cre+). c-Fos cells are localized closer to the subgranular zone (SGZ) in the Cre+ group. Calibration bar, 100 μm. ( G ) c-Fos+ density in the CA3 region of the hippocampus in each of the groups of mice. No significant difference was observed in any of the groups (one-way ANOVA, F (2,13) = 2.27, p = 0.143; Tukey post hoc test indicates that there is no significant difference between any pairs of groups). ( H ) Blade distribution of c-Fos+ mGCs in the IB and SB of the dorsal DG in mice performing the LS separation receiving either DCZ or DMSO 30 min prior to task performance. Active neurons are distributed preferentially to the SB than the IB in the DG for the control groups. However, this distribution bias is significantly reduced in the Cre+ DCZ group (two-way ANOVA (unbalanced) (blade: F (1,24) = 659.55, p < 2.2 × 10⁻¹⁶; group: F (2,24) = 3.48 × 10⁻¹³, p = 1; group × blade: F (1,16) = 0.040, p = 0.845). Tukey post hoc test. Significant differences were observed for IB comparisons: Cre+ DCZ vs Cre− DCZ (p = 0.000548) and Cre+ DCZ vs Cre+ DMSO (p = 0.00765). Significant differences were observed for SB comparisons: Cre+ DCZ vs Cre− DCZ (p = 0.000548) and Cre+ DCZ vs Cre+ DMSO (p = 0.00765)). ( I ) Spatial distribution of c-Fos+ GCs along hilar to molecular layer axes of the SB (0–120 μm) and the IB (0–120 μm) of the dorsal DG, with 0 μm indicating the hilar position. c-Fos+ cells are localized closer to the SGZ in the Cre+ DCZ group. ( J ) Cartoon illustrating the DG with its two blades: IB and SB. Dentate granule cells are depicted in gray, while red circles represent activity labeled cells in the DG. For Cre+ DCZ mice, the distribution of labeled neurons is closer to SGZ, away from outer radial granule cell layer (GCL) and more evenly distributed in IB and SB.

Article Snippet: Strain, strain background ( Mus musculus ) , R26-LSL-Gi-DREADD (hM4Di) , Jackson Laboratory , JAX 026219 , Chemogenetic inhibition model.

Techniques: Immunohistochemical staining, Labeling, Comparison, Immunohistochemistry, Control, Activity Assay