Journal: bioRxiv

Article Title: Oxygen chemoreceptor inhibition by dopamine D 2 receptors in isolated zebrafish gills

doi: 10.1101/2024.10.08.617247

Figure Lengend Snippet: (A) Addition of SQ22536, an adenylyl cyclase (AC) inhibitor, decreased the effect of hypoxia on intracellular Ca 2+ . (B) Summary data as treated in (A) showing reduction in average (±S.E.M.) F/F 0 (Kruskal-Wallis test, P=0.0099, n=6). (C) Forskolin, an AC activator, partially recovered the suppressive effect of dopamine on the Ca 2+ response to hypoxia. (D) Summary data as treated in (C) showing recovery in average (±S.E.M.) F/F 0 of the hypoxic response from dopamine with forskolin (Kruskal-Wallis test, P=0.0019, n=6). The response to hypoxia fully recovered following both treatments (Kruskal-Wallis test, P>0.99, n=6).

Article Snippet: To identify an intracellular mechanism for D 2 -receptors, the AC inhibitor SQ22536 (cat. no 1435, Tocris) and AC activator forskolin (cat. no. 11018, Cayman Chemical) were tested.

Techniques:

Journal: bioRxiv

Article Title: Adrenergic signaling gates astrocyte responsiveness to neurotransmitters and control of neuronal activity

doi: 10.1101/2024.09.23.614537

Figure Lengend Snippet: ( A ) Schematic showing hypothesized mechanism of gating via the G-protein signaling downstream of Oct-TyrR. ( B ) Mean trace (thick line) and 95% confidence interval (thin lines) of cAMP levels as measured by the FRET ratio of the cAMP indicator cAMPFIRE-H in astrocytes (n=5). Tyramine exposure causes a decrease in cAMP levels which aligns with its hypothesized function as a G ⍺i GPCR and this decrease is prevented by knocking down Galphai . ( C ) Quantification of astrocyte calcium response demonstrates that the gating of all NTs is dependent on G ⍺i signaling but cAMP modulation is only sufficient to gate dopamine. ( D ) Example traces of astrocytes treated with the adenylyl cyclase inhibitor SQ22536 responding to dopamine but not glutamate. ( E ) Quantification of astrocyte calcium responses shows that dopamine gating is dependent on Dop2R but not Dop1R1 . Dopamine gating can also be achieved by first treating astrocytes with CMPD101 – which inhibits kinase-mediated internalization of receptors – and this gating is similarly dependent on Dop2R. ( F ) Example traces of astrocytes responding to dopamine but not glutamate following exposure to CMPD101. ( G ) Schematic showing the hypothesized mechanism that links tyramine exposure to dopamine gating. ( H ) Traces (without TTX) of Ddc + dopamine neuron activity. Bath application of dopamine normally inhibits Ddc + neuron activity but becomes excitatory after pre-exposure to CMPD101. Knocking down Dop2R in astrocytes reverts the effects of CMPD101 and bath application of dopamine becomes inhibitory once again. ( I ) Quantification of neuronal activity from experiments outlined in 2H. ( J ) Schematic showing hypothesized role of astrocyte gating of Dop2R on the response of dopaminergic neurons to bath application of dopamine ( K ) Diagram of larval righting assay. ( L ) Larval crawling analysis shows that Dop2R and Krz manipulations in astrocytes do not affect baseline larval locomotion. ( M ) Quantification of latency to right of larvae turned to their posterior side (righting). Knocking down Dop2R in astrocytes with or without Gal80 expression in neurons leads to slower righting. Further, krz knockdown and overexpression have bidirectional effects on larval righting that align with the hypothesized role of Krz in internalizing Dop2R. * Indicates p-value < 0.05; details of statistical comparisons and exact p-values in . All error bars represent SEM. Red dots in bar graphs correspond to traces chosen as example.

Article Snippet: Sq22536 (Tocris, 1435) was used at a concentration of 50 μM and Drosophila tissue and astrocyte cultures were exposed to the drug for 10 min to allow for sufficient inhibition of adenylyl cyclase before proceeding with experiments.

Techniques: Activity Assay, Expressing, Knockdown, Over Expression

Journal: bioRxiv

Article Title: Adrenergic signaling gates astrocyte responsiveness to neurotransmitters and control of neuronal activity

doi: 10.1101/2024.09.23.614537

Figure Lengend Snippet: ( A ) Schematic showing method of calcium imaging in primary rat astrocytes. ( B ) Example image of primary rat astrocytes loaded with the calcium indicator Fluo-4. (Scale bar = 20 μm) ( C ) Schematic of NE and drug selectivity for ⍺1 vs ⍺2 adrenergic receptors. ( D ) Example traces of rat astrocyte calcium responses. Both NE and the ⍺2 adrenergic agonist UK14304 can gate the response to dopamine. ( E ) Quantification of astrocyte calcium responses to various drugs and dopamine suggest that the cellular mechanism of gating is shared between Drosophila and mammalian astrocytes. Namely, dopamine gating can be mediated by G ⍺i adrenergic stimulation, cAMP modulation, and CMPD101-mediated inhibition of internalization. NE exposure can also gate glutamate and there is a trend towards NE gating acetylcholine ( F ) Example astrocyte calcium responses to SQ22536 + dopamine but not dopamine alone shown via pseudo colored Fluo-4 fluorescence. (Scale bar = 20 μm) ( G ) Example trace of rat astrocytes responding to dopamine following SQ22536 mediated inhibition of adenylyl cyclase but not to dopamine alone nor to dopamine following adenylyl cyclase activation via forskolin ( H ) Example images of astrocytes (phase) with or without treatment with CMPD101 for 30 minutes and then stained with an antibody against the extracellular domain of DRD2 (DRD2 ED; green) (Scale bar = 10 μm; brightness of the DRD2 ED staining enhanced in the example control image to demonstrate that astrocyte staining is at or near background levels). ( I ) Quantification of primary rat astrocytes stained for the extracellular domain of DRD2 shows that manipulations that can gate the dopamine response in Drosophila and rat also lead to increased externalization of DRD2. * Indicates p-value < 0.05; details of statistical comparisons and exact p-values in . All error bars represent SEM. Red dots correspond to traces chosen as example.

Article Snippet: Sq22536 (Tocris, 1435) was used at a concentration of 50 μM and Drosophila tissue and astrocyte cultures were exposed to the drug for 10 min to allow for sufficient inhibition of adenylyl cyclase before proceeding with experiments.

Techniques: Imaging, Inhibition, Fluorescence, Activation Assay, Staining, Control

Journal: bioRxiv

Article Title: The Orphan G Protein-Coupled Receptor GPR52 is a Novel Regulator of Breast Cancer Multicellular Organization

doi: 10.1101/2024.07.22.604482

Figure Lengend Snippet: (A) CAMs that were differentially expressed in GPR52 KO MDA-MB-468 cells based on RNA-sequencing. P-value<0.05. n=3. (B) KMplot breast cancer RNA-seq webtool overall survival curves for patients with low versus high MCAM mRNA expression in resected tumors for all breast cancer subtypes. Low versus high cutoff was determined based on the maximum segregation between the groups. (C) TNMplot correlation of MCAM and GPR52 transcript expression in breast tumors from an RNA-sequencing dataset. (D) MDA-MB-468 cells were cultured in monolayer (TC-treated, non-PDL coated) and treated with forskolin (FSK), SQ22536 (ACi), or vehicle control for 24 hours. Cell lysates were probed for MCAM and beta-actin.

Article Snippet: Forskolin (Santa Cruz Biotechnology #SC-3562) and SQ22536 (Santa Cruz Biotechnology #SC-201572) were resuspended in ethanol at 1 mM and DMSO at 14mM, respectively, and frozen.

Techniques: RNA Sequencing, Expressing, Cell Culture, Control