Journal: The Journal of Neuroscience

Article Title: A Novel CCK Receptor GPR173 Mediates Potentiation of GABAergic Inhibition

doi: 10.1523/JNEUROSCI.2035-22.2023

Figure Lengend Snippet: Reagents, cell lines, mice and oligonucleotides

Article Snippet: The following transgenic mice were used: Vgat-ires-Cre ( Slc32a1tm2(Cre)Lowl /J, Vgat-Cre, C57 background, The Jackson Laboratory), mCCK-cKO (CCK-cKO, C57BL/6J background, Cyagen), Vgat-Cre-CCK-cKO (cross of Vgat-Cre and CCK-cKO mice to specifically knock out CCK in GABAergic neurons), CCK-ires-Cre (Cck tm1.1(Cre)Zjh /J, C57BL/6J background; for short CCK-Cre, The Jackson Laboratory), PV-ires-Cre (Pvalb tm1(cre)Arbr /J, PV-Cre, C57BL/6J background, The Jackson Laboratory), CCK-CreER (Cck tm2.1(Cre/ERT2)Zjh /J, CCK-KO, C57BL/6J background, The Jackson Laboratory), CCK-AR/BR-KO (Cckar tm1Kpn Cckbr tm1Kpn /J, CCK1R/2R-KO, 129S1 background, The Jackson Laboratory), and C57BL/6J mice.

Techniques: Imaging, Stable Transfection, Expressing

Journal: The Journal of Neuroscience

Article Title: A Novel CCK Receptor GPR173 Mediates Potentiation of GABAergic Inhibition

doi: 10.1523/JNEUROSCI.2035-22.2023

Figure Lengend Snippet: Known CCK receptors do not mediate CCK signaling in CCK-INs. A , Schematic drawings show rAAV9-mDlx-DIO-ChR2-mCherry-WPRE-pA (mDlx-DIO-ChR2-mCherry) and AAV9-mDlx-Cre-WPRE-pA (mDlx-Cre) injected into CCK1R/2R-KO or CCK-KO mice. B , AAV9-infected mCherry neurons colabeled with GAD67 (left, CCK1R/2R-KO mouse; right, CCK-KO mouse). Scale bars: 50 and 10 µm, respectively. Right, Bar chart shows the percentage of mCherry neurons colabeled with GAD67 in CCK1R/2R-KO and CCK-KO mice. C , Schematic drawings show in vitro patch-clamp recordings from pyramidal neurons while delivering a laser pulse to activate infected GABAergic neurons in CCK-KO and CCK1R/2R-KO mice. Neuronal responses to a step current confirmed that the recorded neurons were excitatory. D , Traces of IPSCs in response to the laser pulse in CCK1R/2R-KO (top) and CCK-KO (bottom) mice before and after HFLS of infected GABAergic neurons. E , Sampled neuronal responses to HFLS in CCK1R/2R-KO and CCK-KO mice. F , Time course of normalized IPSCs before and after (for 25 min) HFLS in CCK-KO (black, n = 9 neurons; one-way ANOVA with Tukey's post hoc test, N.S., not significant) and CCK1R/2R-KO mice (green, n = 9 neurons; one-way ANOVA with Tukey's post hoc test, **** p < 0.0001; two-way RM ANOVA with Tukey's post hoc test, #### p < 0.0001). G , Schematic illustration of the setup for in vivo recordings with laser stimulation in the AC and a natural AS (a noise burst; SOM, PV neuron, PC) in CCK1R/2R-KO (left) and CCK-KO (right) mice. Bottom, Example waveforms from the two groups. H , Normalized LFP response amplitudes to the AS with a preceding laser pulse before and after HFLS in CCK1R/2R-KO (green, n = 7 recording sites, N = 5 mice) and CCK-KO (black, n = 8 recording sites, N = 6 mice; two-way RM ANOVA with Tukey's post hoc test, **** p < 0.0001) mice. Data are expressed as mean ± SEM.

Article Snippet: The following transgenic mice were used: Vgat-ires-Cre ( Slc32a1tm2(Cre)Lowl /J, Vgat-Cre, C57 background, The Jackson Laboratory), mCCK-cKO (CCK-cKO, C57BL/6J background, Cyagen), Vgat-Cre-CCK-cKO (cross of Vgat-Cre and CCK-cKO mice to specifically knock out CCK in GABAergic neurons), CCK-ires-Cre (Cck tm1.1(Cre)Zjh /J, C57BL/6J background; for short CCK-Cre, The Jackson Laboratory), PV-ires-Cre (Pvalb tm1(cre)Arbr /J, PV-Cre, C57BL/6J background, The Jackson Laboratory), CCK-CreER (Cck tm2.1(Cre/ERT2)Zjh /J, CCK-KO, C57BL/6J background, The Jackson Laboratory), CCK-AR/BR-KO (Cckar tm1Kpn Cckbr tm1Kpn /J, CCK1R/2R-KO, 129S1 background, The Jackson Laboratory), and C57BL/6J mice.

Techniques: Injection, Infection, In Vitro, Patch Clamp, In Vivo

Journal: The Journal of Neuroscience

Article Title: A Novel CCK Receptor GPR173 Mediates Potentiation of GABAergic Inhibition

doi: 10.1523/JNEUROSCI.2035-22.2023

Figure Lengend Snippet: Bioinformatics and cell surface binding assay screening for a novel CCK receptor. A , Bar chart showing the highest similarity scores between GPCRs and known CCK receptors (CCK1R and CCK2R) considering the secondary structure in amino acid sequences, including six GPCRs for 5-hydroxytryptamine (serotonin; HTR2C, HTR1F, HTR2B, HTR1E, HTR1B, and HTR1D), an orphan receptor (GPR173), neuropeptide Y receptor Y5 (NPY5R), and adrenergic receptor beta 1 (ADRB1). B–D , Schematic of cell surface binding assay for Flag-tagged GPCRs with negative control (HA-(ε-Ahx) 2 ) or HA-(ε-Ahx) 2 -CCK variants (HA-CCK4, HA-CCK8ns, and HA-CCK8s). Confocal images ( B , top; scale bar, 10 µm) and quantification of fluorescence intensity ratio (F anti-HA /F anti-Flag ; B , D , bottom) in the cell surface binding assay for Flag-CCK2R, Flag-GPR173, Flag-HTR1B, and five other Flag-GPCRs as shown in C . The number of total cells from three independent experiments is defined as n ; n = 33–40, one-way ANOVA with Tukey's post hoc test or unpaired Student's t test, ** p < 0.01, **** p < 0.0001, N.S., not significant. Data are expressed as mean ± SEM.

Article Snippet: The following transgenic mice were used: Vgat-ires-Cre ( Slc32a1tm2(Cre)Lowl /J, Vgat-Cre, C57 background, The Jackson Laboratory), mCCK-cKO (CCK-cKO, C57BL/6J background, Cyagen), Vgat-Cre-CCK-cKO (cross of Vgat-Cre and CCK-cKO mice to specifically knock out CCK in GABAergic neurons), CCK-ires-Cre (Cck tm1.1(Cre)Zjh /J, C57BL/6J background; for short CCK-Cre, The Jackson Laboratory), PV-ires-Cre (Pvalb tm1(cre)Arbr /J, PV-Cre, C57BL/6J background, The Jackson Laboratory), CCK-CreER (Cck tm2.1(Cre/ERT2)Zjh /J, CCK-KO, C57BL/6J background, The Jackson Laboratory), CCK-AR/BR-KO (Cckar tm1Kpn Cckbr tm1Kpn /J, CCK1R/2R-KO, 129S1 background, The Jackson Laboratory), and C57BL/6J mice.

Techniques: Binding Assay, Negative Control, Fluorescence

Journal: The Journal of Neuroscience

Article Title: A Novel CCK Receptor GPR173 Mediates Potentiation of GABAergic Inhibition

doi: 10.1523/JNEUROSCI.2035-22.2023

Figure Lengend Snippet: Bioinformatics and histology screening for a novel CCK receptor. A , Percentage identity correlation matrix of orphan receptors with CCK1R/2R. B , Left, Illustration of mDlx-DIO-ChR2-mCherry injected into the AC of CCK-Cre mice. Middle, Immunochemistry staining images of candidate receptors in CCK-GABAergic synapses. Scale bars: 5 µm. Expanded images are from the white rectangles (top right). The white arrows indicate the colocalization of GPR173 with CCK-GABAergic synapses. Scale bars: 1 µm. C , Bar chart shows numbers of overlay dots [bottom right, mCherry + , Syp (synaptophysin) + , and GPCR + , per mm 2 ; n = 3 independent experiments; one-way ANOVA with Tukey's post hoc test, **** p < 0.0001]. D , Confocal images of Flag-GPR173-transfected HEK293T cells colabeled with anti-GPR173, anti-Flag, and DAPI. Scale bar, 10 µm. Quantitative analysis of colocalization of Flag and GPR173 across the dotted line (labeled in confocal images). The bar chart depicts the percentage of colabeled cells positive for Flag and GPR173 from Flag-GPR173–transfected HEK293T cells stained with anti-GPR173, anti-Flag, and DAPI ( n = 21 cells). E , Super-resolution image of colocalized GPR173 with CCK-GABAergic synapses, GABA A R with CCK-GABAergic synapses, and GABA A R and GPR173 (left). Scale bar, 1 µm. The values for PCC and MOC indicate the colocalization possibility of mCherry with GPR173, mCherry with GABA A R, and GABA A R with GPR173. F , Immunochemistry images of costaining of GPR173 with CaMKII (top) or GAD67 (bottom) in C57BL/6J mice. Scale bar, 50 µm. Group data from colocalization analysis of GPR173 with CaMKII ( n = 11 sections, N = 3 mice) or GAD 67 ( n = 10 sections, N = 3 mice). G , Immunochemistry images for colocalized GPR173 with CCK-GABAergic synapses contacting pyramidal cells and box plot showing numbers of colocalized GPR173 and CCK-GABAergic contacts per pyramidal cell ( n = 49 neurons, 5.69 ± 0.38). Scale bar, 5 µm. Data are expressed as mean ± SEM.

Article Snippet: The following transgenic mice were used: Vgat-ires-Cre ( Slc32a1tm2(Cre)Lowl /J, Vgat-Cre, C57 background, The Jackson Laboratory), mCCK-cKO (CCK-cKO, C57BL/6J background, Cyagen), Vgat-Cre-CCK-cKO (cross of Vgat-Cre and CCK-cKO mice to specifically knock out CCK in GABAergic neurons), CCK-ires-Cre (Cck tm1.1(Cre)Zjh /J, C57BL/6J background; for short CCK-Cre, The Jackson Laboratory), PV-ires-Cre (Pvalb tm1(cre)Arbr /J, PV-Cre, C57BL/6J background, The Jackson Laboratory), CCK-CreER (Cck tm2.1(Cre/ERT2)Zjh /J, CCK-KO, C57BL/6J background, The Jackson Laboratory), CCK-AR/BR-KO (Cckar tm1Kpn Cckbr tm1Kpn /J, CCK1R/2R-KO, 129S1 background, The Jackson Laboratory), and C57BL/6J mice.

Techniques: Injection, Staining, Transfection, Labeling

Journal: The Journal of Neuroscience

Article Title: A Novel CCK Receptor GPR173 Mediates Potentiation of GABAergic Inhibition

doi: 10.1523/JNEUROSCI.2035-22.2023

Figure Lengend Snippet: CCK8s could trigger intracellular Ca 2+ mobilization through GPR173 in CHO-GPR173 cells. A , Schematic illustration of Ca 2+ imaging assay in CHO-GPCR cell lines. B , Dose-dependent Ca 2+ responses of CHO-CCK1R (left; EC 50 = 1.72 ± 1.11 n m ) and CHO-CCK2R (right; EC 50 = 4.64 ± 2.37 n m ) cells provoked by CCK8s. C , EC 50 curve of CCK8s for the CHO-GPR173 cell line (EC 50 = 3.23 ± 1.94 n m ). D , Dose-dependent Ca 2+ response of CHO-GPCR cells provoked by the CCK2R agonist CCK4. E , Dose-dependent Ca 2+ response of CHO-GPCR cells provoked by the CCK1R-specific agonist A-71 623. F , Schematic illustration of knockdown of GPR173 expression in CHO-GPR173 cells using an shRNA of Gpr173. G , 200 n m CCK8s provoked the Ca 2+ response in the scramble or shRNA-infected CHO-GPR173 cells and Gpr173 mRNA expression level in the scramble or shRNA-infected CHO-GPR173 cells. H , Ca 2+ signal responses of CHO-GPR173 cells treated with 10 μ m PNX or 200 n m CCK8s. Relative fluorescence unit (RFU) is defined as the ratio of RFU before and 30 s after adding CCK8s/HHBS (unpaired Student's t test, * p < 0.05,**** p < 0.0001, N.S., not significant). I , Illustration of the β-arrestin1/2 recruitment assay design. J , Dose-dependent response of CCK8s-induced β-arrestin recruitment to CCK1Rs determined by the β-arrestin2 recruitment and β-arrestin1/2 recruitment assays. The two control groups were without GPCR plasmid transfection in HTLA cells. K , L , Dose-dependent response of CCK8s-induced β-arrestin recruitment to CCK2Rs ( K ) and GPR173 ( L ) determined by the β-arrestin2 recruitment and β-arrestin1/2 recruitment assays (at least n = 3 independent experiments; one-way ANOVA with Tukey's post hoc test or unpaired Student's t test, **** p < 0.0001). Data are expressed as mean ± SEM.

Article Snippet: The following transgenic mice were used: Vgat-ires-Cre ( Slc32a1tm2(Cre)Lowl /J, Vgat-Cre, C57 background, The Jackson Laboratory), mCCK-cKO (CCK-cKO, C57BL/6J background, Cyagen), Vgat-Cre-CCK-cKO (cross of Vgat-Cre and CCK-cKO mice to specifically knock out CCK in GABAergic neurons), CCK-ires-Cre (Cck tm1.1(Cre)Zjh /J, C57BL/6J background; for short CCK-Cre, The Jackson Laboratory), PV-ires-Cre (Pvalb tm1(cre)Arbr /J, PV-Cre, C57BL/6J background, The Jackson Laboratory), CCK-CreER (Cck tm2.1(Cre/ERT2)Zjh /J, CCK-KO, C57BL/6J background, The Jackson Laboratory), CCK-AR/BR-KO (Cckar tm1Kpn Cckbr tm1Kpn /J, CCK1R/2R-KO, 129S1 background, The Jackson Laboratory), and C57BL/6J mice.

Techniques: Imaging, Expressing, shRNA, Infection, Fluorescence, Plasmid Preparation, Transfection