Journal: Molecular Psychiatry

Article Title: SSRIs target prefrontal to raphe circuits during development modulating synaptic connectivity and emotional behavior

doi: 10.1038/s41380-018-0260-9

Figure Lengend Snippet: Lack of SERT increases the number of functional PFC-to-DRN synapses. a rAAV-CAG-hChR2(H134R)-mCherry was bilaterally injected into the PFC of P4–P5 control or SERT-KO mice. Photograph showing mCherry expression after the PFC AAV injection (upper left). Optogenetic stimulation and electrophysiological patch clamp recordings were made starting at P28 in coronal sections containing the DRN, as shown by the photograph of the immunolabeling of PFC mCherry+ axons innervating to DRN 5-HT neurons, identified by the presence of the enzyme TPH2 (upper right). b Amplitude of optogenetically evoked EPSCs (oEPSCs) at synapses from PFC terminals onto DRN putative 5-HT neurons (left) and non-5-HT neurons (right) at various light stimulation intensities. In control (SERT Cre/+ ) (5-HT: n = 10 cells/5 animals; non-5-HT: n = 7 cells/4 animals); in SERT-KO (SERT Cre/Cre ) (5-HT: n = 10 cells/3 animals; non-5-HT: n = 6 cells/3 animals). Top: example traces at 9.8 mW (black/gray) and at 2 mW (red) stimulation); Bottom: input/output curves. Two-way ANOVA on 9.8 mW intensity: genotype x cell-type interaction (F 1,29 = 0.003, p = 0.95); Genotype main effect (F 1,29 = 9.32, * p < 0.01); Cell-type main effect (F 1,29 = 0.51, p = 0.48). c AMPAR/NMDAR ratios at synapses from PFC-to-DRN 5-HT neurons (left) and non-5-HT neurons (right) in control (5-HT: n = 10 cells/4 animals; non-5-HT: n = 7 cells/3 animals), and SERT-KO (5-HT: n = 11 cells/3 animals; non-5-HT: n = 6 cells; 3 animals). The AMPAR responses were calculated at the peak of −50 mV, whereas NMDAR responses were determined at + 40 mV, 50 ms after stimulation. Top: example traces; bottom: bar graphs. Two-ways ANOVA: Genotype x Cell-type interaction (F 1,30 = 0.007, p = 0.94); Genotype main effect (F 1,30 = 0.16, p = 0.69); Cell-type main effect (F 1,30 = 4.51, p < 0.05). Blue bars indicate blue light stimulation. Error bars represent SEM

Article Snippet: Subsequently, after a few hours of fixation in 4% PFA at 4 °C, brain slices were processed for immunohistochemistry using a rabbit polyclonal antibody against TPH2 (1:2000, Novus Biologicals, NB100-74555) to identify 5-HT neurons.

Techniques: Functional Assay, Injection, Control, Expressing, Patch Clamp, Immunolabeling

Journal: bioRxiv

Article Title: DNA 5-methylcytosine regulates genome-wide formation of G-quadruplex structures

doi: 10.1101/2023.02.16.528796

Figure Lengend Snippet: Effect of the reduced methylation level on the formation of the G4 structure. (A, E) Detection of methylation levels by immunofluorescence (IF) when cells were treated with 5-aza-dC and RG108. (B, F) The variation in G4 formation detected by IF using BG4 in the cells treated with 5-aza-dC and RG108. (C, D) Quantitative statistical analysis of the methylation level and G4 formation in the cells treated with 5-aza-dC. (G, H) Quantitative statistics of the methylation level and G4 formation in the cells treated with RG108. (I) Design of the knockout target sites and sequencing confirmation of the identified mutant strains. (J, K) Detection of the mRNA and protein levels of the DNMT1 gene in the wild type and mutants. (L-O) Detection and quantitative analysis of methylation levels (L and M) and the G4 signals (N and O) of the wild type and the mutants. (P) Detection of DNMT1 expression by western blot. (Q) The change in the methylation level when DNMT1 was compensatorily expressed in DNMT1-KO-2 cells. (R) Detection of the G4 signal when DNMT1 was compensatorily expressed in DNMT1-KO-2 cells. (S) Quantitative analysis of the immunofluorescence staining in R. Data are the mean ± SD (C, D, G, H, M, O and S) or mean ± SEM (J and Q). Statistical significance was determined by Student’s t test, **p<0.01, ***p<0.001. The scale bars equal 1 μm.

Article Snippet: The compounds 5-aza-dC (5-Aza-2’-deoxycytidine, A3656, Sigma, Shanghai, China) and RG108 (N-phthalyl-L-tryptophan, HY-13642, MedChemExpress, NJ, USA) were dissolved in DMSO (dimethyl sulfoxide).

Techniques: Methylation, Immunofluorescence, Knock-Out, Sequencing, Mutagenesis, Expressing, Western Blot, Staining

Journal: Journal of Clinical Investigation

Article Title: A neuronal action of sirtuin 1 suppresses bone mass in young and aging mice

doi: 10.1172/jci152868

Figure Lengend Snippet: Figure 3. Decreased serotonin synthesis and increased Dbh and MAO-A expression in the brain of TgSirt1 mice. (A) Tph2 expression levels in the brain stem (BS) of 3-month-old TgSirt1 mice (n = 5) versus WT controls (n = 5). (B) 5HT levels in brain stem of TgSirt1 mice (n = 6) versus WT controls (n = 4) measured by HPLC. (C) MAO-A expression levels in the rest of brain (ROB) of 3-month-old TgSirt1 mice (n = 4) versus WT controls (n = 4). (D) MAO-A activity (103 RLU/μg protein/h) in the hypothalamus, brain stem, and rest of brain of TgSirt1 mice treated with phenelzine (n = 5) versus vehi- cle controls (n = 5). (E) 5HT and 5-HIAA levels in brain stem and rest of brain of TgSirt1 mice treated with phenelzine (n = 5) versus vehicle controls (n = 5) measured by HPLC. (F) BV/TV (%); (G) N.Ob/T.Ar (/mm2); (H) BFR/BS (μm3/μm2/yr); and (I) Oc.S/BS (%) of 3-month-old TgSirt1 mice treated with phenelzine (n = 5) versus vehicle (n = 6) and WT controls (n = 5). (J) Representative images of spines from 3-month-old TgSirt1 mice treated with phenelzine versus vehicle and WT controls stained with von Kossa. (K) Dbh expression levels in midbrain (MB) of 3-month-old TgSirt1 mice (n = 5) versus WT controls (n = 5). (L) Tph2 expression levels in brain stem of 3-month-old Sirt1brain –/– mice (n = 4) versus Sirt1COIN/COIN controls (n = 4). (M) MAO-A expression levels in rest of brain of 3-month-old Sirt1brain –/– mice (n = 4) versus Sirt1COIN/COIN controls (n = 4). (N) Dbh expression levels in MB of 3-month-old Sirt1brain –/– mice (n = 4) versus Sirt1COIN/COIN controls (n = 4). Data are represented as mean ± SEM. (A–E and K–N) *P < 0.05, Student’s t test. (F–I) *P < 0.05, TgSirt1 mice treated with phenelzine versus vehicle by 1-way ANOVA.

Article Snippet: Antibodies used were as follows: anti-GFP mouse monoclonal antibody (Takara, catalog 632375); anti-Sirt1 rabbit antibody (Cell Signaling Technology, catalog 2028S); anti-Tph2 rabbit polyclonal antibody (Novus NB, catalog 100-74555); Alexa Fluor 488 AffiniPure donkey anti-mouse IgG (Jackson ImmunoResearch, catalog 715-545-150); and Alexa Fluor 594 AffiniPure donkey, anti-rabbit IgG (Jackson ImmunoResearch, catalog 711-585-152).

Techniques: Expressing, Activity Assay, Staining

Journal: Journal of Clinical Investigation

Article Title: A neuronal action of sirtuin 1 suppresses bone mass in young and aging mice

doi: 10.1172/jci152868

Figure Lengend Snippet: Figure 5. Neuronal SIRT1 decreases bone mass by decreasing serotonin synthesis and enhancing its catabolism through its actions on serotonergic and MAO-A–expressing neurons. (A) Ucp1 expression in BAT of Sirt1Syn –/– mice (n = 7) versus controls (n = 7). (B) Expression of SNS target genes in long bone of Sirt1Syn –/– mice (n = 5) versus controls (n = 5). (C) Tph2 expression in brain stem of Sirt1Syn –/– mice (n = 7) versus controls (n = 7). (D) MAO-A expression and (E) MAO-A activity in rest of brain of Sirt1Syn –/– mice (n = 5) versus controls (n = 5). (F) Dbh expression in MB of Sirt1Syn –/– mice (n = 5) versus controls (n = 5). (G) Bche expression in hypothalamus of Sirt1Syn –/– mice (n = 6) versus controls (n = 6). (H) Ucp1 expression in BAT of Sirt1Sert –/– mice (n = 4) versus controls (n = 8). (I) Expression of SNS target genes in long bone of Sirt1Sert –/– mice (n = 4) versus controls (n = 4). (J) Tph2 expression in brain stem of Sirt1Sert –/– mice (n = 4) versus controls (n = 6). (K) MAO-A expression in rest of brain of Sirt1Sert –/– mice (n = 4) versus controls (n = 8). (L) MAO-A activity in rest of brain of Sirt1Sert –/– mice (n = 5) versus controls (n = 5). (M) Dbh expression in MB of Sirt1Sert –/– mice (n = 4) versus controls (n = 5). (N) Bche expression in hypothalamus of Sirt1Sert –/– mice(n = 4) versus controls (n = 4). (O) Ucp1 expression in BAT of Sirt1Dbh –/– mice (n = 5) versus controls (n = 5). (P) Expression of SNS target genes in long bone of Sirt1Dbh –/– mice (n = 5) versus controls (n = 5). (Q) Tph2 expression in brain stem of Sirt1Dbh –/– mice (n = 4) versus controls (n = 5). (R) MAO-A expression in rest of brain of Sirt1Dbh –/– mice (n = 4) versus controls (n = 5). (S) MAO-A activity in rest of brain of Sirt1Dbh –/– mice (n = 5) versus controls (n = 5). (T) Dbh expression in MB and (U) Bche expression in hypothalamus of Sirt1Dbh –/– (n = 4) versus controls (n = 5). Data are represented as mean ± SEM. *P < 0.05 versus Sirt1COIN/COIN by Student’s t test.

Article Snippet: Antibodies used were as follows: anti-GFP mouse monoclonal antibody (Takara, catalog 632375); anti-Sirt1 rabbit antibody (Cell Signaling Technology, catalog 2028S); anti-Tph2 rabbit polyclonal antibody (Novus NB, catalog 100-74555); Alexa Fluor 488 AffiniPure donkey anti-mouse IgG (Jackson ImmunoResearch, catalog 715-545-150); and Alexa Fluor 594 AffiniPure donkey, anti-rabbit IgG (Jackson ImmunoResearch, catalog 711-585-152).

Techniques: Expressing, Activity Assay