pg97-269  (Bachem)

Journal: Frontiers in Cellular Neuroscience

Article Title: VPAC Receptor Subtypes Tune Purinergic Neuron-to-Glia Communication in the Murine Submucosal Plexus

doi: 10.3389/fncel.2017.00118

Figure Lengend Snippet: VIP-evoked glial and neuronal responses in the presence of various antagonists. (A) The selective VPAC1 antagonist (PG 97-269; 1 μM), the VPAC2 antagonist (PG 99-465; 1 μM), tetrodotoxin (TTX; 1 μM) and the nicotinic antagonist hexamethonium (Hex.; 200 μM) all significantly inhibited the peak amplitude of the VIP (100 μM)-evoked neuronal [Ca 2+ ] i transients (one-way analysis of variance (ANOVA) and Dunnett’s test, *** P < 0.001, **** P < 0.0001). (B) The VPAC2 antagonist also inhibited the number of neurons responding to VIP compared to control. TTX (1 μM) significantly inhibited the number of neurons responding to VIP and near abolished the neuronal response (one-way ANOVA and Dunnett’s test, * P < 0.05, *** P < 0.001). VIP-induced glial [Ca 2+ ] i transients were revealed by antagonists. The number of submucosal glia responding to a local spritz-application of VIP (100 μM) in the presence of (C) the VPAC2 antagonist PG 99-465 (1 μM; n = 7) or tetrodotoxin (TTX; Na + channel blocker; 1 μM; n = 8) was significantly increased compared to control conditions (two-way ANOVA and Sidak’s test, * P < 0.05, ** P < 0.01). Glial responses were not observed in time control experiments where VIP was applied twice under control conditions, separated by a 5 min washout ( n = 25 preparations).

Article Snippet: VIP antagonists used include the VPAC1 antagonist PG97-269, and the VPAC2 antagonist, PG 99-465 (both from Mimotopes).

Techniques: Control

Journal: Frontiers in Cellular Neuroscience

Article Title: VPAC Receptor Subtypes Tune Purinergic Neuron-to-Glia Communication in the Murine Submucosal Plexus

doi: 10.3389/fncel.2017.00118

Figure Lengend Snippet: VPAC1-induced [Ca 2+ ] i transients in enteric neurons and glia. (A) Number of submucosal neurons and glia responding to three repeated local spritz-applications of VPAC1 agonist ([K15, R16, L27]VIP(1-7)/GRF(8-27); 100 μM) separated by 5 min in time control experiments. (B) Maximum amplitudes of VPAC1 agonist-evoked glial [Ca 2+ ] i transients were reproducible over time control experiments. (C) Glial fibers in a submucosal ganglion responding to VPAC1 agonist over time, as indicated by arrows. Scale bar = 20 μm. (D) VPAC1 agonist-induced [Ca 2+ ] i responses of selected glial fibers as indicated by color-coded arrows in (C) The black arrow in the graph marks the application of VPAC1-agonist. (E) Responding glial fibers identified by glial fibrillary acidic protein (GFAP) immunofluorescent labeling.

Article Snippet: VIP antagonists used include the VPAC1 antagonist PG97-269, and the VPAC2 antagonist, PG 99-465 (both from Mimotopes).

Techniques: Control, Labeling

Journal: Frontiers in Cellular Neuroscience

Article Title: VPAC Receptor Subtypes Tune Purinergic Neuron-to-Glia Communication in the Murine Submucosal Plexus

doi: 10.3389/fncel.2017.00118

Figure Lengend Snippet: VPAC1 agonist-evoked glial and neuronal [Ca 2+ ] i responses in the presence of antagonists. (A) The peak amplitude of the VPAC1 agonist ([K15, R16, L27]VIP(1-7)/GRF(8-27); 100 μM)-evoked response was significantly inhibited by the selective VPAC1 antagonist (PG 97-269; 1 μM; one-way ANOVA and Dunnett’s test, ** P < 0.01), but was not affected by TTX (1 μM). The P2 antagonist pyridoxal-phosphate-6-azophenyl-2’,4’-disulfonic acid (PPADS; 30 μM) and the selective P2Y1 antagonist MRS2179 (10 μM) were also effective in inhibiting the VPAC1 agonist response amplitude (one-way ANOVA and Dunnett’s test, * P < 0.05, ** P < 0.01). (B) Only the VPAC1 antagonist significantly reduced the number of glia responding to the VPAC1 agonist (one-way ANOVA and Dunnett’s test, * P < 0.05).

Article Snippet: VIP antagonists used include the VPAC1 antagonist PG97-269, and the VPAC2 antagonist, PG 99-465 (both from Mimotopes).

Techniques:

Journal: Frontiers in Cellular Neuroscience

Article Title: VPAC Receptor Subtypes Tune Purinergic Neuron-to-Glia Communication in the Murine Submucosal Plexus

doi: 10.3389/fncel.2017.00118

Figure Lengend Snippet: Localizing VPAC1 receptor (VPAC1R)-expression in the submucosal plexus of mouse jejunum. (A–D) Confocal micrographs demonstrating that VPAC1R-immunoreactive cell bodies were cholinergic, as marked by (B) ChAT and (C) Hu staining and indicated by the arrow. (E–H) VPAC1R expressing fibers also overlapped with peripherin + nerve fibers (indicated by filled arrowheads). (I–L) However, VPAC1R did not colocalize with the GFAP + glial fibers (open arrowheads). (A–L) are maximum projections of stacks of confocal images. (M–O) Confocal images taken at a single optical plane showing that VPAC1R- and Vesicular acetyltransferase (VAChT)-immunoreactivity also did not overlap (open arrowheads). Scale bars = 20 μm.

Article Snippet: VIP antagonists used include the VPAC1 antagonist PG97-269, and the VPAC2 antagonist, PG 99-465 (both from Mimotopes).

Techniques: Expressing, Staining

Journal: Frontiers in Cellular Neuroscience

Article Title: VPAC Receptor Subtypes Tune Purinergic Neuron-to-Glia Communication in the Murine Submucosal Plexus

doi: 10.3389/fncel.2017.00118

Figure Lengend Snippet: Confocal images of VPAC1 receptor (VPAC1R)-immunoreactive nerve fibers and varicosities in the submucosal plexus of mouse jejunum taken at a single optical plane. (A–C) VPAC1R did not colocalize with tyrosine hydroxylase (TH), as indicated by open arrowheads. (D–F) Confocal images of VPAC1 receptor (VPAC1R)- and calcitonin related-gene peptide (CGRP)-immunoreactive varicosities in a submucosal ganglion. Some overlap between VPAC1R- and CGRP-labeling was observed (filled arrowhead). However, not all VPAC1R + varicosities were CGRP + and vice versa (open arrowhead). Scale bars = 20 μm.

Article Snippet: VIP antagonists used include the VPAC1 antagonist PG97-269, and the VPAC2 antagonist, PG 99-465 (both from Mimotopes).

Techniques: Labeling

Journal: Frontiers in Cellular Neuroscience

Article Title: VPAC Receptor Subtypes Tune Purinergic Neuron-to-Glia Communication in the Murine Submucosal Plexus

doi: 10.3389/fncel.2017.00118

Figure Lengend Snippet: P2Y1 agonist 2-methyl-thio-ADP (2MeSADP)-evoked glial [Ca 2+ ] i responses. (A) The P2Y1 antagonist MRS2179 (10 μM) significantly inhibited the amplitude of glial cells responding to 2MeSADP (100 μM; one-way ANOVA and Dunnett’s test, * P < 0.05). (B) The number of glia responding to 2MeSADP was increased in the presence of TTX (1 μM) compared to control (one-way ANOVA and Dunnett’s test, * P < 0.05). (C–F) Some glial cells displayed [Ca 2+ ] i transients in response to both 2MeSADP (100 μM) and VPAC1 agonist ([K15, R16, L27]VIP(1-7)/GRF(8-27); 100 μM), as indicated by arrows. However, the latency of the 2MeSADP-evoked glial [Ca 2+ ] i responses was significantly shorter than that of the VPAC1 agonist. The black arrows in the graphs mark the application of agonist.

Article Snippet: VIP antagonists used include the VPAC1 antagonist PG97-269, and the VPAC2 antagonist, PG 99-465 (both from Mimotopes).

Techniques: Control

Journal: Frontiers in Cellular Neuroscience

Article Title: VPAC Receptor Subtypes Tune Purinergic Neuron-to-Glia Communication in the Murine Submucosal Plexus

doi: 10.3389/fncel.2017.00118

Figure Lengend Snippet: A schematic of VIP-activated pathways in the mouse submucosal plexus determined using Ca 2+ imaging . This wiring diagram represents the most plausible neuro-glia circuit based on our experimental observations. VIP acting via VPAC1 and/or VPAC2 receptors elicits [Ca 2+ ] i transients in submucosal neurons. Neurons respond directly to VIP or secondarily via nicotinic transmission. Selective activation of VPAC1 receptors evokes glial [Ca 2+ ] i transients. This neuron to glia signaling pathway involves a TTX-insensitive mechanism. VPAC1 receptors expressed on cholinergic neurons and/or on CGRP + nerve terminals may be involved. VIP also activates a VPAC2 receptor-mediated pathway that inhibits glial [Ca 2+ ] i responses. However, whether the VPAC2 receptor-expressing neuron inhibits glial responses directly or by inhibiting the VPAC1 receptor-mediated pathway, is yet to be determined. Note that the neurochemical identities of neurons involved are not illustrated for simplicity. Further, the possibility that VPAC1- and VPAC2-receptors are expressed on the same neuron, but activate different intracellular signaling pathways, cannot be excluded.

Article Snippet: VIP antagonists used include the VPAC1 antagonist PG97-269, and the VPAC2 antagonist, PG 99-465 (both from Mimotopes).

Techniques: Imaging, Transmission Assay, Activation Assay, Expressing, Protein-Protein interactions