Journal: medRxiv

Article Title: Intraluminal Prucalopride Increases Propulsive Motor Activities in the Human Colon

doi: 10.1101/2020.05.24.20111930

Figure Lengend Snippet: 5-HT and 5-HT 4 R positive cells were completely co-localized in human colonic epithelium of all sections.

Article Snippet: Primary antibodies were polyclonal rabbit anti-5-HT 4 receptor (Atlas Antibodies, Cat # HPA040591, Lot # R36821, 1:200) and monoclonal mouse anti-5-HT (ThermoFisher Scientific, Cat # MA5-12111, 1:100); secondary antibodies were Alexa 488 goat anti-rabbit IgG (Jackson ImmunoResearch, 1:300) for 5-HT4R staining and Cy3 goat anti-mouse IgG (Jackson ImmunoResearch, 1:200) for 5-HT staining.

Techniques:

Journal: Glia

Article Title: Serotonin receptor 4 regulates hippocampal astrocyte morphology and function.

doi: 10.1002/glia.23933

Figure Lengend Snippet: FIGURE 1 Astrocytes express the 5-HT4R in vivo. (a) Expression of the 5-HT4R in S100β-positive cells throughout the mouse brain shown by immunohistochemical staining. (b) Magnification of hippocampal structures shown in (a) revealed expression of the 5-HT4R in hippocampal astrocytes. Scale bar 50 μm. (c) Split channels for indicated area in (b). White arrows exemplarily point to 5-HT4R expressing astrocytes. Scale bar 50 μm. (d) 3D representation of a hippocampal astrocyte visualized by S100β and 5-HT4R staining. 5-HT4R was heterogeneously distributed. The grey arrow points towards a branch with less 5-HT4R staining than the one marked by the white arrow. Scale bar 50 μm. (e) STED microscopy images showed clustering of 5-HT4R on S100β-positive cells. The dotted white line represents the astrocyte outline identified by S100β labeling. Scale bar 2 μm. (f) Magnification of an astrocyte process shown in (e). Scale bar 1 μm [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Following antibodies were used for staining: S100β (mouse, dilution 1:500, #66028, Abcam, Cambridge, UK), 5-HT4R (rabbit, dilution 1:500, #ASR-036, Alomone Labs, Jerusalem, Israel), don- key anti-mouse Alexa Fluor 488 (dilution 1:400, #715-545-150, Jackson ImmunoResearch, Ely, UK), goat anti-rabbit Alexa Fluor 546 (dilution 1:400, #A-11035, Invitrogen, Carlsbad, CA), donkey anti-mouse DyLight 649 (dilution 1:400, #715-495-151, Jackson ImmunoResearch, Ely, UK), and goat anti-rabbit Cyanine3 (dilution 1:400, #A10520 Invitrogen, Carlsbad, CA).

Techniques: In Vivo, Expressing, Immunohistochemical staining, Staining, Microscopy, Labeling

Journal: Glia

Article Title: Serotonin receptor 4 regulates hippocampal astrocyte morphology and function.

doi: 10.1002/glia.23933

Figure Lengend Snippet: FIGURE 2 Cultured astrocytes as a model to investigate 5-HT4R signaling. (a) Immunocytochemical labeling of 5-HT4R astrocyte marker GFAP-positive cells in primary hippocampal cultures from WT mice. No signal of the 5-HT4R was detected in astrocytes from 5-HT4R-ko animals. (b) Visualization of 5-HT4R and S100β protein in cultured astrocytes from WT and 5-HT4R-ko mice. Scale bars in (a) and (b) 50 μm (left overview) and 20 μm (right magnification). (c) Schematic illustration of established 5-HT4R signaling pathways. Upon activation, the 5-HT4R induced signaling via GαS or Gα13 heterotrimeric G proteins, leading to cAMP upregulation via adenylyl cyclase (AC) or RhoA activation and subsequent actin cytoskeleton reorganization, respectively. (d) Relative mRNA expression levels of 5-HT4R and its down-stream effectors in primary hippocampal astrocyte cultures from WT and 5-HT4R-ko mice. Statistical significance was evaluated using unpaired two-tailed t test, N = 4 independent cultures [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Following antibodies were used for staining: S100β (mouse, dilution 1:500, #66028, Abcam, Cambridge, UK), 5-HT4R (rabbit, dilution 1:500, #ASR-036, Alomone Labs, Jerusalem, Israel), don- key anti-mouse Alexa Fluor 488 (dilution 1:400, #715-545-150, Jackson ImmunoResearch, Ely, UK), goat anti-rabbit Alexa Fluor 546 (dilution 1:400, #A-11035, Invitrogen, Carlsbad, CA), donkey anti-mouse DyLight 649 (dilution 1:400, #715-495-151, Jackson ImmunoResearch, Ely, UK), and goat anti-rabbit Cyanine3 (dilution 1:400, #A10520 Invitrogen, Carlsbad, CA).

Techniques: Cell Culture, Labeling, Marker, Protein-Protein interactions, Activation Assay, Expressing, Two Tailed Test

Journal: Glia

Article Title: Serotonin receptor 4 regulates hippocampal astrocyte morphology and function.

doi: 10.1002/glia.23933

Figure Lengend Snippet: FIGURE 4 5-HT4R-activation leads to actin reorganization. (a) Representative images showing F-actin fraction in fixed hippocampal astrocytes. In WT astrocytes the stimulation with BIMU8 (30 min, 10 μM) resulted in an increase in the F-actin fraction. In 5-HT4R-ko astrocytes the initial fraction was higher and addition of BIMU8 had no effect. The cells from the excerpts indicated by white boxes are additionally shown uniformly in grey color on the right, to emphasize the cell area and branching. Scale bar 20 μm. (b) The quantification reflected an increase in the F- to G-actin ratio upon BIMU8 treatment in WT but not in 5-HT4R-ko astrocytes. Statistical differences were calculated using one-way ANOVA with Dunnett's multiple comparisons post hoc test, n ≥25 cells, N = 3. (c) Border-distance plots indicate the gradient of F- and G-actin intensities from the outer cell border to the center with a proportional shift upon BIMU8 treatment in WT (upper plot) but not in 5-HT4R-ko astrocytes (lower plot), n ≥25 cells, N = 3. (d) Sholl analysis reflected a reduction in arborization complexity in BIMU8 treated astrocytes (Area under the curve [AUC] 224 ± 15) compared with control cells (AUC 270 ± 15). Unpaired t-test with Welch's correction, p = .038, n ≥31 cells, N = 3. Arrows point towards direction of change [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Following antibodies were used for staining: S100β (mouse, dilution 1:500, #66028, Abcam, Cambridge, UK), 5-HT4R (rabbit, dilution 1:500, #ASR-036, Alomone Labs, Jerusalem, Israel), don- key anti-mouse Alexa Fluor 488 (dilution 1:400, #715-545-150, Jackson ImmunoResearch, Ely, UK), goat anti-rabbit Alexa Fluor 546 (dilution 1:400, #A-11035, Invitrogen, Carlsbad, CA), donkey anti-mouse DyLight 649 (dilution 1:400, #715-495-151, Jackson ImmunoResearch, Ely, UK), and goat anti-rabbit Cyanine3 (dilution 1:400, #A10520 Invitrogen, Carlsbad, CA).

Techniques: Activation Assay, Control

Journal: Glia

Article Title: Serotonin receptor 4 regulates hippocampal astrocyte morphology and function.

doi: 10.1002/glia.23933

Figure Lengend Snippet: FIGURE 5 Impact of 5-HT4R activation on the actin cytoskeleton is G protein dependent. (a) Representative images showing F-actin fraction in cultured hippocampal astrocytes infected with AAVs encoding shRNA scramble, shRNA against Gα13 or shRNA against GαS proteins. Stimulation with 5-HT4R agonist BIMU8 (30 min, 10 μM) increased the F-actin fraction under control conditions as well as in cells expressing less GαS. If Gα13 expression was downregulated, this effect was reversed and stimulation of the receptor led to restoration of levels similar to the control. The cells from the excerpts indicated by white boxes are additionally shown uniformly in grey color on the right, to emphasize the cell area and branching. Scale bar 20 μm. (b) Quantification of the F-actin fraction. Statistical differences were calculated using two-way ANOVA with Tukey's multiple comparisons post hoc test, n ≥101 cells, N = 3. (c) Sholl analysis revealed a reduction in astrocyte complexity upon BIMU8 stimulation. Knockdown of GαS by shRNA enhanced this effect, while stimulation had the opposite effect in astrocytes with downregulated Gα13 protein levels, leading to increased complexity. Two-way ANOVA with Tukey's multiple comparisons test. shRNA-scr and shRNA-scr + BIMU8: radius 18 μm p = .0195; shRNA-Gα13 and shRNA-Gα13 + BIMU8: radius 70 μm p = .0045; shRNA-GαS and shRNA-GαS + BIMU8: radius 46 μm p = .0007; n ≥25 cells, N = 3. Arrows point out direction of change upon BIMU8 treatment. (d) Quantification of astrocyte size (2D) showed the impact of G protein signaling in regulation of cell size. Astrocytes occupied more space when expressing shRNA against Gα13 compared with astrocytes expressing shRNA against GαS. This difference was more prominent upon stimulation with 5-HT4R agonist BIMU8. Statistical differences were calculated using two-way ANOVA with Tukey's multiple comparisons post hoc test, n ≥98 cells, N = 3 [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Following antibodies were used for staining: S100β (mouse, dilution 1:500, #66028, Abcam, Cambridge, UK), 5-HT4R (rabbit, dilution 1:500, #ASR-036, Alomone Labs, Jerusalem, Israel), don- key anti-mouse Alexa Fluor 488 (dilution 1:400, #715-545-150, Jackson ImmunoResearch, Ely, UK), goat anti-rabbit Alexa Fluor 546 (dilution 1:400, #A-11035, Invitrogen, Carlsbad, CA), donkey anti-mouse DyLight 649 (dilution 1:400, #715-495-151, Jackson ImmunoResearch, Ely, UK), and goat anti-rabbit Cyanine3 (dilution 1:400, #A10520 Invitrogen, Carlsbad, CA).

Techniques: Activation Assay, Cell Culture, Infection, shRNA, Control, Expressing, Knockdown

Journal: Glia

Article Title: Serotonin receptor 4 regulates hippocampal astrocyte morphology and function.

doi: 10.1002/glia.23933

Figure Lengend Snippet: FIGURE 6 5-HT4R activation in astrocytes impacts neuronal signaling. (a) Illustration of the experimental setup with three conditions. 5-HT4R-ko mice were used for preparation of mixed hippocampal cultures (HCC) and the receptor was either rescued in astrocytes or neurons using viral infection of AAV-mGFAP-5-HT4R-eGFP or AAV-syn-5-HT4R-eGFP, respectively. In a third (control) condition cells were infected with AAV-tdTomato. Representative traces of electrophysiological recordings of neurons in those cultures are shown on the right for all three conditions. Only rescued expression of the 5-HT4R in astrocytes, but not neurons, influenced mEPSC frequency after BIMU8 stimulation. (b) Quantification of mEPSC frequency in hippocampal neurons after application of 10 μM 5-HT4R agonist BIMU8. Increase of mEPSC frequency was only present if astrocytes expressed 5-HT4Rs, but not if the receptor was only present in neurons. No change was observed upon BIMU8 stimulation in a 5-HT4R-ko culture or when RhoA signaling was blocked by cell-permeable, highly potent ROCK inhibitor Y-27632 (50 μM). Statistical analysis was performed using two-way ANOVA with Sidak's multiple comparisons post hoc test, n ≥7 [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Following antibodies were used for staining: S100β (mouse, dilution 1:500, #66028, Abcam, Cambridge, UK), 5-HT4R (rabbit, dilution 1:500, #ASR-036, Alomone Labs, Jerusalem, Israel), don- key anti-mouse Alexa Fluor 488 (dilution 1:400, #715-545-150, Jackson ImmunoResearch, Ely, UK), goat anti-rabbit Alexa Fluor 546 (dilution 1:400, #A-11035, Invitrogen, Carlsbad, CA), donkey anti-mouse DyLight 649 (dilution 1:400, #715-495-151, Jackson ImmunoResearch, Ely, UK), and goat anti-rabbit Cyanine3 (dilution 1:400, #A10520 Invitrogen, Carlsbad, CA).

Techniques: Activation Assay, Infection, Control, Expressing

Journal: Glia

Article Title: Serotonin receptor 4 regulates hippocampal astrocyte morphology and function.

doi: 10.1002/glia.23933

Figure Lengend Snippet: FIGURE 7 Astrocytic 5-HT4R impact neuronal properties in vivo. (a) Schematic overview of acute slice recordings. 5-HT4R-ko mice were stereotactically injected with AAV-GFAP-5-HT4R-eGFP and AAV-GFAP-tdTomato into separate hemispheres to selectively restore astroglial 5-HT4Rs in the hippocampus. After 3 weeks acute slices were subjected to electrophysiological investigations. fEPSPs were evoked by electrical stimulation of CA3–CA1 Schaffer collaterals and recorded in the stratum radiatum (S.R.) of the CA1 region. (b) Astroglial expression of AAVs was visualized using two-photon excitation fluorescence microscopy. Transfected astrocytes were uniformly distributed across stratum oriens (S.O.) and S.R. Unlike cytosolic tdTomato the 5-HT4R-eGFP fusion protein was located to astrocytic membranes. Stratum pyramidale (S.P.) indicated for orientation. (c) Representative fEPSPs recorded in S.R. with gradually increasing stimulation intensities (left panel, examples for 50, 100, 200 μA). fEPSP slopes recorded near 5-HT4R-eGFP expressing astrocytes (green) or areas covered by tdTomato expressing astrocytes (red) were not statistically different (right panel, p = .87, two-way repeated measures ANOVA, eGFP n = 11, tdTomato n = 14). (d) Representative pairs of fEPSPs recorded with an inter-stimulus interval of 25 ms (left panel). The paired-pulse ratio (PPR) obtained at short inter-stimulus intervals (ISI) was lower when astroglial 5-HT4R expression was rescued compared with tdTomato-positive control slices (p = .0084, two-way repeated measures ANOVA, eGFP n = 10, tdTomato n = 14, Tukey post hoc test: 25 ms, p = .0065, **50 ms, p = .145). (e–f) The experiments were repeated in the presence of ROCK inhibitor Y-27632 (20 μM). The fEPSP slopes recorded near 5-HT4R-eGFP-expressing astrocytes were not significantly different from fEPSPs recorded close to tdTomato-expressing astrocytes (p = .225, two-way repeated measures ANOVA, eGFP n = 13, tdTomato n = 12). Also the PPR was not different between these conditions when ROCK was blocked (p = .873, two-way repeated measures ANOVA, eGFP n = 13, tdTomato n = 12) [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Following antibodies were used for staining: S100β (mouse, dilution 1:500, #66028, Abcam, Cambridge, UK), 5-HT4R (rabbit, dilution 1:500, #ASR-036, Alomone Labs, Jerusalem, Israel), don- key anti-mouse Alexa Fluor 488 (dilution 1:400, #715-545-150, Jackson ImmunoResearch, Ely, UK), goat anti-rabbit Alexa Fluor 546 (dilution 1:400, #A-11035, Invitrogen, Carlsbad, CA), donkey anti-mouse DyLight 649 (dilution 1:400, #715-495-151, Jackson ImmunoResearch, Ely, UK), and goat anti-rabbit Cyanine3 (dilution 1:400, #A10520 Invitrogen, Carlsbad, CA).

Techniques: In Vivo, Injection, Expressing, Fluorescence, Microscopy, Transfection, Positive Control