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: 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

Journal: Journal of Clinical Investigation

Article Title: Melanocortin 4 receptor stimulation prevents antidepressant-associated weight gain in mice caused by long-term fluoxetine exposure

doi: 10.1172/jci151976

Figure Lengend Snippet: Figure 8. Mechanisms of Flx-induced short-term and long-term hyperphagia and weight gain. In the short term (left), within hours of oral treatment with Flx, Tph2+Htr1a+ DRN neurons are inhibited, leading to a decrease in the serotonin-dependent activation of POMC+Htr2c+ ARC (ARCPOMC/Htr2c) and Mc4r+ PVN (PVNMc4r) neurons, which causes an increase in food intake (left, top). Blocking the Htr1a-dependent inhibition of Tph2+Htr1a+DRN (DRNTph2/Htr1a) neurons using Prop (left, middle) or activating POMC+Htr2c+ ARC (ARCPOMC/Htr2c) neurons using Lorca (left, bottom) can therefore normalize feeding in this setting. Upon long-term treatment (right), however, Flx decreases Htr2c expression and signaling and inhibits STAT3 phosphorylation in ARC neurons, resulting in reduced α-MSH production (right, top). This noncanonical and multifactorial activity of Flx explains the paradoxical hyperphagia and weight gain associated with its long-term use as well as the failure of Lorca to counter this effect. In contrast, cotreatment with Lcn2, a Mc4r ligand, can normal- ize feeding and prevent weight gain (right, bottom).

Article Snippet: The following primary antibodies were used for immunohistochemical analyses: rabbit c-Fos at 1:1000 (Cell Signaling Technology 2250, RRID:AB_2247211); guinea pig c-Fos at 1:1000 (Synaptic Systems, 226 004, RRID:AB_2619946); rabbit Tph2 at 1:1000 (Novus, NB100-74555, RRID:AB_1049988); rabbit POMC at 1:4000 (Phoenix Pharmaceuticals, H-029-30, RRID:AB_2307442); sheep α-MSH at 1:10,000 (MilliporeSigma, AB5087, RRID:AB_91683); rabbit NPY at 1:1000 (Abcam, ab221145); and mouse Htr2c at 1:200 (Santa Cruz 1 3J Clin Invest.

Techniques: Activation Assay, Blocking Assay, Inhibition, Expressing, Phospho-proteomics, Activity Assay

Journal: Neuropsychopharmacology

Article Title: Adult AMPA GLUA1 Receptor Subunit Loss in 5-HT Neurons Results in a Specific Anxiety-Phenotype with Evidence for Dysregulation of 5-HT Neuronal Activity

doi: 10.1038/npp.2014.332

Figure Lengend Snippet: Immunohistochemical verification of 5-HT neuron-specific GLUA1 loss. Brains of Gria15-HT−/− and Gria1fl/fl mice were removed 4 weeks after tamoxifen administration. Double-fluorescent IHC with TPH2- (green) and GLUA1-antibodies (red) was performed, as shown in color-separated images and in the corresponding overlay (a3–f3) in the dorsal (a1–d3) and median (e1–f3) raphe nucleus. White boxes indicate the tissue areas enlarged in the insets. (a1–b3, e1–e3) In Gria1fl/fl mice, most immunopositive cells for TPH2 in both the median and dorsal raphe nucleus co-expressed GLUA1. Labeling for TPH2 was distributed in cell bodies and dendrites, while labeling for GLUA1 was mostly restricted to the neuropil but also found in the cytoplasm of neurons (f.i. white arrows in b1–b3 and e1–e3). (c1–d3, f1–f3) In Gria15-HT−/− mice, most TPH2-immunopositive cells in both the median and dorsal raphe nucleus were immunonegative for GLUA1. Labeling for TPH2 was distributed in cell bodies and dendrites, while labeling for GLUA1 was absent from cell bodies (f.i. white asterisks in d2) and only detectable in the neuropil. Scale bar: a, c, e, f: 100 μm; b, d: 50 μm.

Article Snippet: The following antibodies were used for IHC: rabbit anti-TPH2 (Dianova; 1 : 1000) mouse anti-GLUA1 (NeuroMab; 1 : 400).

Techniques: Immunohistochemical staining, Labeling

Journal: Neuropsychopharmacology

Article Title: Adult AMPA GLUA1 Receptor Subunit Loss in 5-HT Neurons Results in a Specific Anxiety-Phenotype with Evidence for Dysregulation of 5-HT Neuronal Activity

doi: 10.1038/npp.2014.332

Figure Lengend Snippet: Selective reduction of TPH2-expression in raphe nuclei. Brains of Gria1fl/fl and Gria15-HT−/− mice were dissected for analysis of TPH2 expression by immunoblotting. (a) Representative Western blots. TPH2 antibody stainings of lysates from midbrain raphe nuclei (RN), hippocampus (HC) and prefrontal cortex (PFC) revealed immunoreactive bands above 50 kDa. As a loading control, the same lysates were stained with actin antibodies yielding immunoreactive bands at roughly 40 kDa with the same intensities. (b) Brain-region specific TPH2 expression. Analysis of TPH2 expression revealed a significant reduction of TPH2-dependent fluorescence in raphe lysates by 14% in Gria15-HT−/− mice (n=5; p<0.05). In contrast, lysates from the projection areas of 5-HT neurons (HC and PFC) did not show significant differences in fluorescence intensities between Gria1fl/fl and Gria15-HT−/− mice.

Article Snippet: The following antibodies were used for IHC: rabbit anti-TPH2 (Dianova; 1 : 1000) mouse anti-GLUA1 (NeuroMab; 1 : 400).

Techniques: Expressing, Western Blot, Staining, Fluorescence