Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Serotonin functions as a bidirectional guidance molecule regulating growth cone motility

doi: 10.1007/s00018-020-03628-2

Figure Lengend Snippet: 5-HT1b and 5-HT2a receptors are expressed in growth cones and filopodia. a–j Sensory neuron growth cones immunostained for serotonin receptors, 5-HT1b (green in a, red in g), 5-HT2a (green, d and h) as well as filamentous actin to highlight the extent of the growth cone (Phalloidin, red; b and e). The receptors 5-HT1b and 5HT2a were observed as a widespread punctate distribution throughout the growth cone (g–h). Punctate expression was evident in filopodia (j) with isolated 5-HT1b (green, *), 5-HT2a (red, #) as well as colocalised (arrowheads) puncta on filopodial shafts. There was prominent colocalization of 5-HT2a and 5-HT1b receptors in peripheral areas (i, arrow). Scale bars are 5 μm (a–i) and 1 μm (j). k Turning responses to serotonin were sensitive to chlorpromazine. Attractive turning of growth cones to 5HT-Lo (n = 7, p = <0.0001) and repulsion to 5HT-Hi (n = 8, p = 0.003) were significantly different when cultures were treated with chlorpromazine. l Axon extension was not significantly different in any experimental treatments (ns, p > 0.05). (Mann–Whitney u-test)

Article Snippet: DRG sensory neurons were fixed with paraformaldehyde (PFA) (4%, Sigma-Aldrich) in phosphate buffered saline (PBS) overnight at 4 °C, washed in PBS (3 × 10 min), for some experiments, cells were permeabilised for 1 h with blocking solution (PBS containing 0.4% Triton X-100; 5% goat serum, Sigma-Aldrich & Gibco) and immunostained with a rabbit antibody to serotonin receptors 5-HT1b (1:500; Abcam) or 5-HT2a (1:500; Alomone Labs) in blocking solution overnight at 4 °C.

Techniques: Expressing, Isolation, MANN-WHITNEY

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Serotonin functions as a bidirectional guidance molecule regulating growth cone motility

doi: 10.1007/s00018-020-03628-2

Figure Lengend Snippet: 5HT-Lo elicits growth cone attraction through the 5-HT2a receptor. a Growth cone attraction was measured in the presence of 5HT-Lo gradients (p = <0.0001) and TCB-2 gradients (p = 0.0002). Growth cone attraction to 5HT-Lo was abolished by pharmacological application of the selective 5-HT2a receptor antagonist, ritanserin (n = 17, p = 0.0001) and the PLC inhibitor, U-73122 (n = 9, p = 0.0018). b Growth cone attraction to 5HT-Lo was not perturbed when growth cones treated with a specific 5-HT1b antagonist, GR55562 (n = 8, p = <0.0001), or the 5-HT3 antagonist ondansetron (n = 8, p = 0.0082). Depletion of ER calcium with thapsigargin (n = 19, p = 0.0003) abolished attractive turning to 5HT-Lo, while inhibition of VGCCs with nifedipine (n = 15, p = 0.0330) had no significant effect. Turning angles were compared to vehicle and 5HT-Lo and only significant differences shown in (a) and (b). Kruskal–Wallis, Dunn’s multiple comparison test. c, d Axon extension was not significantly different in any experimental treatments (ns, p > 0.05). Kruskal–Wallis, Dunn’s multiple comparison test

Article Snippet: DRG sensory neurons were fixed with paraformaldehyde (PFA) (4%, Sigma-Aldrich) in phosphate buffered saline (PBS) overnight at 4 °C, washed in PBS (3 × 10 min), for some experiments, cells were permeabilised for 1 h with blocking solution (PBS containing 0.4% Triton X-100; 5% goat serum, Sigma-Aldrich & Gibco) and immunostained with a rabbit antibody to serotonin receptors 5-HT1b (1:500; Abcam) or 5-HT2a (1:500; Alomone Labs) in blocking solution overnight at 4 °C.

Techniques: Inhibition, Comparison

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Serotonin functions as a bidirectional guidance molecule regulating growth cone motility

doi: 10.1007/s00018-020-03628-2

Figure Lengend Snippet: 5HT-Hi promotes growth cone repulsion through the 5-HT1b receptor. a Growth cone repulsion was measured in the presence of 5HT-Hi gradients (p = <0.0001) and CP-94253 gradients (p = 0.0018). Growth cone repulsion to 5HT-Hi was altered to levels indistinguishable from random growth (vehicle) with application of the selective antagonist to 5-HT1b, GR55562 (n = 16, p = <0.0001), by activating adenylate cyclase with forskolin (n = 8, p = 0.0003) and restoration of cAMP signals with Sp-cAMPs (n = 8, p = 0.0001). b Repulsion to 5HT-Hi was not perturbed when growth cones were treated with the 5-HT2a receptor antagonist ritanserin (n = 8, p = 0.002) or the 5-HT3 antagonist ondansetron (n = 10, p = 0.0003). Inhibition of ER calcium release with thapsigargin (n = 10, p = <0.0001) or calcium influx with nifedipine (n = 8, p = <0.0001) did not alter growth cone repulsion from 5HT-Hi. Turning angles were compared to vehicle and 5HT-Hi. c, d Axon extension was not perturbed by any pharmacological application (ns, p > 0.05). Turning angles were compared to vehicle and 5HT-Hi. (Kruskal–Wallis, Dunn’s multiple comparison test)

Article Snippet: DRG sensory neurons were fixed with paraformaldehyde (PFA) (4%, Sigma-Aldrich) in phosphate buffered saline (PBS) overnight at 4 °C, washed in PBS (3 × 10 min), for some experiments, cells were permeabilised for 1 h with blocking solution (PBS containing 0.4% Triton X-100; 5% goat serum, Sigma-Aldrich & Gibco) and immunostained with a rabbit antibody to serotonin receptors 5-HT1b (1:500; Abcam) or 5-HT2a (1:500; Alomone Labs) in blocking solution overnight at 4 °C.

Techniques: Inhibition, Hi-C, Comparison

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Serotonin functions as a bidirectional guidance molecule regulating growth cone motility

doi: 10.1007/s00018-020-03628-2

Figure Lengend Snippet: 5HT-Lo exposure causes asymmetric distribution of 5-HT receptors to the turning or motile side of growth cones. Schematic a showing growth cone divided into “near” and “far” regions with respect to micropipet position (located on the upper left side). b–d Growth cones immunostained after turning in response to 5HT-Lo and stained for 5-HT2a (green, b), 5-HT1b (red, c) and merged in (d). Dotted line separates the near and far regions of growth cone (near vs far). (e) Representative image of filopodia with 5-HT2a (green), 5-HT1b (red) receptor puncta. (f) There was no significant near/far bias in the number of filopodia following exposure to 5HT-Lo (n = 8, p = 0.9004), 5HT-Hi (n = 13, p = 0.5450) or BDNF (n = 10, p = 0.7907) compared to vehicle. (Data not normally distributed: Kruskal–Wallis, Dunn’s multiple comparison test). g There were significantly more (n = 36, p = 0.0033) 5-HT2a puncta in filopodia on the near side of growth cones exposed to 5HT-Lo compared to all other treatments. h There was no bias in 5-HT1b puncta in filopodia of growth cones. (Mann–Whitney U-test). (i-j) When entire growth cones were analysed, i 5HT-2a (n = 9, p = 0.0006) and j 5HT-1b (n = 9, p = 0.0028) receptor translocation was significantly biased in growth cones exposed to 5HT-Lo. All ratios were compared to vehicle (Data normally distributed: Shapiro–Wilk. One-way ANOVA, Tukey’s multiple comparison test). Scale bars are 5 μm (Fig. 7b–d) and 1 μm (Fig. 7e)

Article Snippet: DRG sensory neurons were fixed with paraformaldehyde (PFA) (4%, Sigma-Aldrich) in phosphate buffered saline (PBS) overnight at 4 °C, washed in PBS (3 × 10 min), for some experiments, cells were permeabilised for 1 h with blocking solution (PBS containing 0.4% Triton X-100; 5% goat serum, Sigma-Aldrich & Gibco) and immunostained with a rabbit antibody to serotonin receptors 5-HT1b (1:500; Abcam) or 5-HT2a (1:500; Alomone Labs) in blocking solution overnight at 4 °C.

Techniques: Staining, Comparison, MANN-WHITNEY, Translocation Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Serotonin functions as a bidirectional guidance molecule regulating growth cone motility

doi: 10.1007/s00018-020-03628-2

Figure Lengend Snippet: 5HT-Lo exposure causes asymmetric distribution of membrane 5-HT2a receptors to the motile side of growth cones. a–h Representative growth cones exposed to vehicle and 5HT-Lo gradients (micropipette on upper left side) and stained for membrane localization of receptors 5-HT1b (green, a, e), 5-HT2a (green, c, g) and merged with actin, (red, b, d, f, h). Dotted line separates the near and far regions of the growth cone. i Increase magnification of representative image h to show the presence of 5-HT2a receptor membrane on most filopodia (*) of the growth cone (yellow *, insets). Scale bars are 5 μm. (j-k)There was no significant near/far bias in the amount of f-actin (/area) (j) and the number of filopodia (k) in growth cones exposed to vehicle and 5HT-Lo. Kruskal–Wallis, Dunn’s multiple comparison post hoc test. (l) There was no significant bias in the total percentage of filopodia containing 5HT2a and 5HT1b puncta (60%). One-way ANOVA followed by Tukey’s multiple comparison post hoc test. (m) There was no significant near/far bias in the amount of 5-HT2a and 5-HT1b puncta per filopodia. (n) Analysis of 5-HT receptor membrane localization showed translocation of 5-HT2a (n = 18, p = <0.0001) to the near side of growth cones exposed to 5HT-Lo while no significant translocation of 5-HT1b (n = 15, p = 0.346) was observed. (Mann–Whitney U-test)

Article Snippet: DRG sensory neurons were fixed with paraformaldehyde (PFA) (4%, Sigma-Aldrich) in phosphate buffered saline (PBS) overnight at 4 °C, washed in PBS (3 × 10 min), for some experiments, cells were permeabilised for 1 h with blocking solution (PBS containing 0.4% Triton X-100; 5% goat serum, Sigma-Aldrich & Gibco) and immunostained with a rabbit antibody to serotonin receptors 5-HT1b (1:500; Abcam) or 5-HT2a (1:500; Alomone Labs) in blocking solution overnight at 4 °C.

Techniques: Membrane, Staining, Comparison, Translocation Assay, MANN-WHITNEY

Journal: Journal of Microbiology and Biotechnology

Article Title: The Effect of Lactobacillus gasseri BNR17 on Postmenopausal Symptoms in Ovariectomized Rats

doi: 10.4014/jmb.2105.05032

Figure Lengend Snippet: Serum estradiol (E2) ( A ), Calcitonin ( B ), Osteocalcin ( C ), and 5-HT-2A ( D ) were measured after 14 weeks of initial administration ( n = 6~8 rats per group). Sophora japonica fruit extract (SJFE) treatment was used to create the positive control.

Article Snippet: The concentrations of estradiol (E2, CSB-E05110r), osteocalcin (CSB-E05129r), calcitonin (CSB-E05132r), estradiol (CSB-E05110r), and Serotonin 2A (5-HT-2A, CSB-E14956r) were assessed using an ELISA kit (CUSABIO, China).

Techniques: Positive Control

Journal: Ecotoxicology and environmental safety

Article Title: Chronic developmental exposure to low-dose ([C8mim][PF6]) induces neurotoxicity and behavioural abnormalities in rats.

doi: 10.1016/j.ecoenv.2021.112806

Figure Lengend Snippet: Fig. 4. Developmental exposure to low-dose ([C8MIM] [PF6]) increased the expression of NMDA receptors. (A) The mRNA level of major receptors (nmdar1, nmdar2a, nmdar2b for NMDA receptor; drd1 and drd2 for dopamine receptor; 5-ht2ar for 5-HT receptor; gabaarα1 for GABA receptor) for main neurotransmitters in hippocampus and prefrontal cortex. (B) The protein levels of NMDA receptors (NMDAR1, NMDAR2A, and NMDAR2B) in the hippocam pus and prefrontal cortex. (C) The protein levels of dopa mine receptors (DRD1 and DRD2), 5-HT receptors (5- HT2AR), and GABA receptors (GABAARα1) in the hippo campus and prefrontal cortex. (D) Statistical analysis of protein expression. n = 3 for each group. Student’s t-test was performed for the analysis. The data are presented as the mean ± SEM. *p＜0.05, **p＜0.01, ***p＜0.001.

Article Snippet: Membranes were treated with 5% skim milk for 1 h, followed by incubation with primary antibodies against NMDAR1 (ab109182, Abcam), NMDAR2A (ab124913, Abcam), NMDAR2B (ab65783, Abcam), DRD1 (ab81296, Abcam), DRD2 (ab30743, Abcam), 5-HT2AR (PB9599, Boster), GABAARα1 (ab252430, Abcam), PSD95 (36233S, Cell Signalling Technology), IBA1 (GTX100042, GeneTex), GFAP (BA0056, Boster), NeuN (ab177487, Abcam), MBP (BA0094, Boster), IFN-γ (PAA033Ra01, Cloud-clone Corp.), IL-1β (PAA563Ra01, Cloud-clone Corp.), TNF-α (PAA133Ra01, cloud-clone corp.), NF-ĸB (66535-1-lg, Proteintech), Histone H3 (AF0009, Beyotime), NRF2 (bs-1074R-1, bioss), PPARγ(ab272718, Abcam), COX2 (ab179800, Abcam), iNOS (ab178945, Abcam), Caspase 3 (9662s, CST), Bax (SC-7480, SANTA CRUZ), Bcl-2 (sc7382, SANTA CRUZ), GAPDH (5174T, CST), and β-actin (3700s, CST) at a 1:1000 dilution overnight at 4 ◦C.

Techniques: Expressing

Journal: Frontiers in Psychiatry

Article Title: Histone deacetylase inhibitors mitigate antipsychotic risperidone-induced motor side effects in aged mice and in a mouse model of Alzheimer’s disease

doi: 10.3389/fpsyt.2022.1020831

Figure Lengend Snippet: Effects of histone deacetylase (HDAC) inhibitors on histone acetylation at the 5htr2a and Drd2 gene promoters in the striatum in young and aged mice. (A) No significant differences of H3K27ac binding at the 5htr2a promoter between young and aged group was found, but a significant increase in H3K27ac binding at the 5ht2a promoter in the striatum was found in aged and young mice co-treated with RIS and VPA or MS-275 compared to those treated with RIS alone. (B) H3K27ac binding at the Drd2 promoter was significantly decreased in aged mice as compared to young mice in Veh treated group. However, H3K27ac binding at the Drd2 promoter in the striatum was observed to be significantly increased in aged mice co-treated with RIS and HDAC inhibitors VPA or MS-275 when compared to those treated with Veh alone. Data are presented as mean ± SEM, ( n = 5). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Blots were blocked for 1-hour with 5% non-fat dry milk (Biorad, 1706404) and incubated using 1:1,000 5HT2A (Boster Biological, PA1373), 1:500 D2R (Millipore, AB5084P), or 1:1000 B-actin (Santa Cruz, sc-47778) overnight at 4°C.

Techniques: Histone Deacetylase Assay, Binding Assay

Journal: Pharmacology, biochemistry, and behavior

Article Title: Chronic methamphetamine self-administration dysregulates 5-HT2A and mGlu2 receptor expression in the rat prefrontal and perirhinal cortex: comparison to chronic phencyclidine and MK-801.

doi: 10.1016/j.pbb.2018.09.007

Figure Lengend Snippet: Identification of antibodies suitable for the detection of 5-HT2A and mGlu2 receptor proteins in the rat brain tissue. (A) Validation of six different commercially-available antibodies in the frontal cortex of rats (R), wild-type mice (WT) and 5-HT2A knockout mice (KO). Only one antibody (Immunostar #24288) detected a ~75kDa band (◀) corresponding to a mature, glycosylated rat 5-HT2A receptor that was not present in 5-HT2A KO mice. (B) Deglycosylation of the wild type (WT) mouse cortical lysate (lanes 2 and 3) by PNGase F increased electrophoretic mobility of the ~75 kDa (◀) band to yield ~52 kDa (◁) band (detected by Immunostar #24288 5-HT2A receptor antibody) that corresponds to the sequenced-based molecular weight of the rat 5-HT2A receptor. For a comparison, the 5-HT2A receptor detection (Immunostar #24288) in the rat (R) cerebral cortex has been shown (lane 1). (C) Immunostaining of rat cortical sections with 5-HT2A #24288 antibody produced a laminar staining pattern consistent with the known 5-HT2A distribution in the mPFC (as shown under 10× magnification). This signal was not present in the mPFC slices, or in the mPFC tissue homogenates incubated with the 5-HT2A antiserum preadsorbed with its immunogenic peptide.

Article Snippet: Only one antibody (Immunostar #24288) detected a ~75kDa band (◀) corresponding to a mature, glycosylated rat 5-HT2A receptor that was not present in 5-HT2A KO mice. (B) Deglycosylation of the wild type (WT) mouse cortical lysate (lanes 2 and 3) by PNGase F increased electrophoretic mobility of the ~75 kDa (◀) band to yield ~52 kDa (◁) band (detected by Immunostar #24288 5-HT2A receptor antibody) that corresponds to the sequenced-based molecular weight of the rat 5-HT2A receptor.

Techniques: Biomarker Discovery, Knock-Out, Molecular Weight, Comparison, Immunostaining, Produced, Staining, Incubation

Journal: The Journal of Neuroscience

Article Title: Dopamine Receptor Agonists Regulate Levels of the Serotonin 5-HT 2A Receptor and its mRNA in a Subpopulation of Rat Striatal Neurons

doi: 10.1523/JNEUROSCI.16-11-03727.1996

Figure Lengend Snippet: Average numbers (per 0.212 mm 2 of tissue section) of neurons labeled with the 5-HT 2A cRNA, double-labeled with the 5-HT 2A and PPE cRNAs, or Nissl-stained on adjacent sections in two sectors of the striatum

Article Snippet: The serotonin 5-HT 2A receptors were labeled with [ 125 I]DOI (DuPont, Billerica, MA; specific activity 2200 Ci/mmol), according to Mengod’s modification ( Mengod et al., 1990 ; Radja et al., 1993 ) of the protocol of McKenna et al. (1989) .

Techniques: Labeling, Control