Journal: eLife

Article Title: The anterior cingulate cortex and its role in controlling contextual fear memory to predatory threats

doi: 10.7554/eLife.67007

Figure Lengend Snippet:

Article Snippet: For the pharmacogenetic inhibition, ACA was injected bilaterally with 150 nl of AAV5-hSyn-HA-hM4D(Gi)-IRES-mCitrine (Dr. Bryan Roth; Addgene plasmid #50464) or AAV5-hSyn-eGFP (titer≥7×1012 vg/ml; Addgene viral prep #50465-AAV5) as control.

Techniques: Transfection, Construct, Virus, Plasmid Preparation, Software

Journal: Neuropsychopharmacology

Article Title: Role of RGS12 in the differential regulation of kappa opioid receptor-dependent signaling and behavior

doi: 10.1038/s41386-019-0423-7

Figure Lengend Snippet: RGS12 expression reduces potency of the KOR agonist U50,488, yet augments agonist-stimulated recruitment of β-arrestin to KOR via independent mechanisms. a-c RGS12 expression reduces potency of the KOR agonist U50,488 to a greater extent than for the MOR agonist DAMGO or the DOR agonist DADLE. GloSensor-22F (Promega) luciferase-based measurements of cAMP levels within HEK293T cells stimulated with 100 nM isoproterenol and simultaneously treated with opioid receptor-selective agonists following transient co-expression of (a) KOR cDNA plus indicated RGS12 expression plasmids, b MOR cDNA with or without wildtype (WT) RGS12, or c DOR cDNA with or without WT RGS12. (a, inset) PTX (200 ng/mL) pretreatment abolished KOR agonist-driven cAMP inhibition, confirming that the assay is dependent on Gi/o-mediated signaling. Wildtype RGS12 expression reduced U50,488 potency by 20-fold, i.e., 6 to 8-fold more than its effects on DAMGO (3-fold) and DADLE (2.5-fold) potency. Loss-of-function point mutants of RGS12 curtailed U50,488 potency reductions from 20-fold down to 3-to-4-fold. Data were normalized to vehicle control conditions and are expressed as the mean ± SEM from multiple experiments (N = 3). Concentration-response curves were fit by four-parameter non-linear regression (Prism 7). d-f The PDZ domain docking site of KOR (-NKPV-c) is necessary, but not sufficient, to completely account for the specificity of RGS12 for KOR over MOR in assays of GPCR-mediated G protein signaling. d Schematic representing creation of KOR expression cDNA containing mutation to the C-terminus (-NKPV-c to -AAAA-c) and MOR expression cDNA containing mutation of its wildtype C-terminus (MOR: -APLP-c) to KOR’s wildtype C-terminus (-NKPV-c). e Mutation to the C-terminus of KOR curtailed RGS12-mediated U50,488 potency reductions from 20-fold (e.g., panel a) to 4-fold. f RGS12 reduced MOR agonist potency by 6-fold following substitution of the MOR C-terminus with the wildtype KOR C-terminus, relative to a 3-fold potency reduction observed with wildtype MOR (e.g., panel b). Data are normalized to vehicle control and expressed as mean ± SEM from N = 3 experiments. Dose-response curves fit by four parameter non-linear regression (Prism). g-j RGS12 expression augments agonist-stimulated recruitment of β-arrestin to KOR, but not MOR nor DOR, in a G protein-independent manner. Tango assays of β-arrestin recruitment within HTLA cells [53, 54] stimulated with indicated agonists following transient co-expression of (g) KOR cDNA plus indicated RGS12 expression plasmids, (h) KOR cDNA with or without WT RGS12 following pretreatment with PTX or vehicle, (i) MOR cDNA with or without WT RGS12, or (j) DOR cDNA with or without WT RGS12. Agonist potency (pEC50 values) did not differ between conditions. U50,488-induced β-arrestin recruitment efficacy was ~3-fold greater when either WT RGS12, or loss-of-function point mutants of RGS12, was co-expressed with KOR, whereas WT RGS12 only increased β-arrestin recruitment to MOR by 1.2-fold and DOR by 1.1-fold. PTX pretreatment did not affect U50,488-induced β-arrestin recruitment to KOR (panel H) in the presence or absence of WT RGS12. Data were normalized to vehicle control conditions (fold change) and are expressed as the mean ± SEM from multiple experiments (N = 3). Curves were analyzed by three-parameter non-linear regression and panel H was analyzed by two-way ANOVA with Sidak’s post hoc test. k [3H]U69,593 saturation binding analysis of vSTR membranes from β-arrestin-2 knockout (βarr2KO) mice and wildtype (WT) controls. l As derived from data in panel K and parallel binding data from dSTR membrane samples, Bmax was increased in vSTR (but not dSTR) of β-arrestin-2 knockout (βarr2KO vSTR Bmax = 150.1 ± 17.1 fmol/mg protein vs wildtype vSTR Bmax = 93.1 ± 14.6 fmol/mg protein; βarr2KO dSTR Bmax = 74.5 ± 9.8 fmol/mg protein vs wildtype dSTR Bmax = 69.3 ± 7.7 fmol/mg protein). The KD for [3H]U69,593 did not differ across genotypes (βarr2KO vSTR: 7.1 ± 1.8 nM; WT vSTR: 8.4 ± 2.9 nM; βarr2KO dSTR: 7.8 ± 2.3 nM; WT dSTR: 6.6 ± 1.7 nM;). Data are the mean ± SEM and panel L was analyzed by two-way ANOVA with Sidak’s post hoc test (n = 12 mice per group) (*p < 0.05)

Article Snippet: HTLA cells expressing β-arrestin-TEV protease fusion and tetracycline transactivator-driven luciferase reporter [ 53 ] were transfected via CaPO 4 [ 52 ] with opioid receptor-Tango vectors (FLAG-KOR-Tango, FLAG-MOR-Tango, or FLAG-DOR-Tango; contributed to Addgene by Dr. Bryan Roth) with or without WT hRGS12, Rgs12 E740K , or Rgs12 R1206F vectors.

Techniques: Expressing, Luciferase, Inhibition, Control, Concentration Assay, Mutagenesis, Binding Assay, Knock-Out, Derivative Assay, Membrane

Journal: Neuropsychopharmacology

Article Title: Role of RGS12 in the differential regulation of kappa opioid receptor-dependent signaling and behavior

doi: 10.1038/s41386-019-0423-7

Figure Lengend Snippet: RGS12 interacts with KOR and RGS12 loss results in elevated KOR sensitivity and binding sites in the vSTR. a Representative immunoblots indicating that KOR and RGS12 co-immunoprecipitate in the vSTR of wildtype mice (N = 2 experiments). Subpanel (i) indicates loss of RGS12 immunoreactivity within whole lysates of ventral striatum from Rgs12-null mice. Subpanel (ii) demonstrates that endogenous KOR protein is co-immunoprecipitated from ventral striatal lysate upon immunoprecipitation of endogenous RGS12 protein. Subpanels (ii) and (iii) indicate that immunoblots probed with a mixture of anti-KOR and an anti-KOR blocking peptide yield no KOR immunoreactivity, confirming antibody specificity. Immunoprecipitation of RGS12 in RGS12-null vSTR results in greatly reduced signal of KOR immunoreactivity (see densitometric quantitation below immunoblot of subpanel (ii)), supporting that KOR immunoreactivity in wildtype IP samples is not due to non-specific pull down. b Co-immunoprecipitation analyses of HEK293T cells transiently transfected with human opioid receptor (with N-terminal HA-tag) and/or full-length RGS12 vector DNA. Robust content of RGS12 is seen in the anti-HA antibody immunoprecipitate when RGS12 is co-expressed with KOR, whereas co-expression of the related opioid receptor MOR yields only non-specific binding signal (i.e., compare first vs last lanes). Parallel immunoblots containing whole cell lysates resolved by SDS-PAGE demonstrate the presence or absence of RGS12, KOR, and/or MOR expression in appropriate conditions. c Total locomotion (over 30 min) by RGS12-null mice and wildtype littermate controls following administration of U50,488. RGS12-null, but not wildtype, mice exhibit a hypolocomotor effect to 2.5 mg/kg U50,488 relative to vehicle controls. At 5 mg/kg U50,488, RGS12-null and wildtype controls both display reduced locomotor activity relative to vehicle controls. Data are the mean ± SEM and tested by two-way ANOVA with Dunnet’s post hoc test (n = 7–13 mice per group) (ns, p > 0.05; *p < 0.05; ***p < 0.001; ****p < 0.0001). d [35S]GTPγS incorporation into vSTR membranes from RGS12-null mice and wildtype littermate controls upon activation with KOR agonist U50,488. Data are normalized to vehicle (saline) control (expressed as fold change). vSTR membranes from RGS12-null mice exhibit increased sensitivity to U50,488 relative to wildtype controls. Inset, [35S]GTPγS binding (at 100 μM U50,488) by dSTR membranes from RGS12-null mice and wildtype littermate controls. All data are the mean ± SEM and tested by two-way ANOVA with Sidak’s post hoc test (n = 13–19 mice per group) (*p < 0.05; ***p < 0.001). e [3H]U69,593 saturation binding analysis of vSTR membranes from RGS12-null (Rgs12−/−) mice and wildtype (Rgs12+/+) littermate controls. Non-specific binding was determined in the presence of 10 μM nor-BNI (KOR antagonist). Inset, As derived from data in panel B and parallel binding data from dSTR membrane samples, Bmax was quantified and seen to be increased in vSTR (but not dSTR) of RGS12-null mice (RGS12-null vSTR Bmax = 62.5 ± 3.9 fmol/mg protein vs wildtype vSTR Bmax = 42.1 ± 3.3 fmol/mg protein). The KD for [3H]U69,593 did not differ across genotypes (RGS12-null vSTR: 1.8 ± 0.4 nM; WT vSTR: 1.8 ± 0.4 nM). Data are the mean ± SEM and tested by two-way ANOVA with Sidak’s post hoc test (n = 9–12 mice per group) (***p < 0.001)

Article Snippet: HTLA cells expressing β-arrestin-TEV protease fusion and tetracycline transactivator-driven luciferase reporter [ 53 ] were transfected via CaPO 4 [ 52 ] with opioid receptor-Tango vectors (FLAG-KOR-Tango, FLAG-MOR-Tango, or FLAG-DOR-Tango; contributed to Addgene by Dr. Bryan Roth) with or without WT hRGS12, Rgs12 E740K , or Rgs12 R1206F vectors.

Techniques: Binding Assay, Western Blot, Immunoprecipitation, Blocking Assay, Quantitation Assay, Transfection, Plasmid Preparation, Expressing, SDS Page, Activity Assay, Activation Assay, Saline, Control, Derivative Assay, Membrane