Journal: Frontiers in Pharmacology

Article Title: Activation of Dopamine D1-D2 Receptor Complex Attenuates Cocaine Reward and Reinstatement of Cocaine-Seeking through Inhibition of DARPP-32, ERK, and ΔFosB

doi: 10.3389/fphar.2017.00924

Figure Lengend Snippet: The effects of D1-D2 heteromer stimulation and inactivation on basal conditioned place preference. (A) Vehicle-conditioned rats did not exhibit a preference toward a particular chamber. D1-D2 heteromer stimulation by SKF 83959 (1.5 mg/kg, s.c.) induced conditioned place aversion (CPA) as the animals spent significantly less time in the drug paired chamber. (B) SKF 83959-induced CPA was abolished by pre-treatment by the D1-D2 heteromer selective disrupting peptide, TAT-D1, but not the control TAT-Sc peptide. (C) Inactivation of D1-D2 heteromer by TAT-D1 resulted in conditioned place preference (CPP) as the rats spent significantly more time in the drug paired chamber, not observed with the control TAT-Sc. (D) Representative western blots (inset) and histogram showing the amount of D1R co-immunoprecipitated with D2R from the NAc of rats treated with saline or SKF 83959. Pretreatment with TAT-D1 led to decreased co-immunoprecipitated receptors. An aliquot of each sample was used as a control for WB (input control). (E,F) The CPA induced by D1-D2 heteromer stimulation was abolished by Cdk5 inhibitor roscovitine pre-treatment (200 nmol, i.c.v, E ) or intra-accumbal injection (30 nmol, F ). (G) Representative western blot and histogram showing the density of Thr75-DARPP-32 phosphorylation (pT75) relative to GAPDH (as loading control). Data represent means ± SEM of n = 8–10 rats/group. ( * p < 0.05, ** p < 0.01: compared to saline).

Article Snippet: Protein homogenates (300 μg /each condition) from rat NAc were incubated with an anti-D2R antibody (Rabbit, Alomone) at 4°C overnight under gentle rotation.

Techniques: Conditioned Place Preference, Western Blot, Immunoprecipitation, Injection

Journal: Frontiers in Pharmacology

Article Title: Activation of Dopamine D1-D2 Receptor Complex Attenuates Cocaine Reward and Reinstatement of Cocaine-Seeking through Inhibition of DARPP-32, ERK, and ΔFosB

doi: 10.3389/fphar.2017.00924

Figure Lengend Snippet: Activation of Thr75-DARRP-32 by D1-D2 heteromer in rat NAc. (A,B) Rats ( n = 8/group) were injected with saline or SKF 83959 (1.5 mg/kg, s.c.), sacrificed 15, 45, or 90 min later, and phospho-Thr34-DARPP-32 (pT34) or phospho-Thr75-DARPP-32 (pT75) analyzed by western blot with GAPDH as loading control. (A) Representative blots of pT34 and pT75. (B) Quantification of blots from all animals represented as % mean ± SEM of control (saline values), ( ** p < 0.05). (C–F) Rats were injected with saline or SKF 83959 (1.5 mg/kg), sacrificed 15 min later, and immunohistochemistry performed using anti-pT34 or anti-pT75 assessed in the three types of MSNs: D1R-only (red arrow), Enk-only (D2R, green arrow) or D1R and ENK (D1-D2 heteromer)-expressing neurons (yellow arrow). (C) Representative confocal images of pT75-DARPP-32. (D) Quantification of pT75-DARPP-32 fluorescence in MSNs ( n = numbers of neurons from at least N = 3 rats/condition). (E) Representative confocal images of pT34-DARPP-32. (F) Quantification of pT34 fluorescence in MSNs. Data represents means ± SEM after removal of non-specific background. ( ** p < 0.01).

Article Snippet: Protein homogenates (300 μg /each condition) from rat NAc were incubated with an anti-D2R antibody (Rabbit, Alomone) at 4°C overnight under gentle rotation.

Techniques: Activation Assay, Injection, Western Blot, Immunohistochemistry, Expressing, Fluorescence

Journal: Frontiers in Pharmacology

Article Title: Activation of Dopamine D1-D2 Receptor Complex Attenuates Cocaine Reward and Reinstatement of Cocaine-Seeking through Inhibition of DARPP-32, ERK, and ΔFosB

doi: 10.3389/fphar.2017.00924

Figure Lengend Snippet: Signaling pathways involved in D1-D2 heteromer modulation of cocaine-induced behaviors: DARPP-32, ERK and ΔFosB. (A) Representative immunoblots of pT34-DARPP-32 (top panel) or pT75-DARPP-32 (lower panel) obtained from NAc of rats conditioned with saline or cocaine (10 mg/kg, i.p.) and injected on the test day with saline or SKF 83959. Loading controls (GAPDH) are shown. Quantification of pT34- and pT75-DARPP-32 immunoblots is shown. (B) Representative immunoblot of pERK44/42 obtained from the NAc of rats conditioned as in (A) is shown. Quantification of pERK44/42 immunoblots obtained from all animals is shown. Results in (A,B) represent the mean ± SEM from 8 to 9 rats/condition. * p < 0.05 and ** p < 0.01 represent significant differences from control. (C) Representative confocal images of pERK assessed in the three types of MSNs: D1R-only (red arrow), Enk-only (D2R, green arrow) or D1R and ENK (D1-D2 heteromer)-expressing neurons (yellow arrow) in MSNs from NAc of saline- or SKF 83959-treated rats. (D) Quantification of pERK fluorescence in MSNs in the NAc. Results are the mean ± SEM of data after removing the non-specific background ( n = number of MSNs from N = at least 3 rats/condition). ( ** p < 0.001; *** p < 0.0001). (E) Representative immunohistochemistry images and their quantification obtained using an antibody against ΔFosB and a secondary antibody conjugated to Alexa-488. Nuclei are stained by DAPI. Rats were treated for 7 days with cocaine (10 mg/kg, i.p.) without or with co-injection of SKF 83959 (1 mg/kg, s.c.). Disrupting the heteromer by repeated injections of TAT-D1 had the same effect as repeated injections of cocaine. Results are means ± SD obtained by the analysis of n = 1,500–1,700 neurons from the NAc of N = 3 rats/condition.

Article Snippet: Protein homogenates (300 μg /each condition) from rat NAc were incubated with an anti-D2R antibody (Rabbit, Alomone) at 4°C overnight under gentle rotation.

Techniques: Western Blot, Injection, Expressing, Fluorescence, Immunohistochemistry, Staining

Journal: Frontiers in Pharmacology

Article Title: Activation of Dopamine D1-D2 Receptor Complex Attenuates Cocaine Reward and Reinstatement of Cocaine-Seeking through Inhibition of DARPP-32, ERK, and ΔFosB

doi: 10.3389/fphar.2017.00924

Figure Lengend Snippet: Evidence for the existence of dopamine D1-D2 receptor heteromer in rat NAc. (A) Proximity ligation assay (PLA) was used to visualize and detect D1R and D2R close proximity. (A1) A scheme depicts the PLA probes used in the present study. (A2–A4) Representative images of PLA signals (red dots) in neurons (nuclei stained by DAPI) in rat caudate putamen (CPu), nucleus accumbens core (NAc-core) and shell (NAc-shell) subregions. (A5) Graph representing the percent of neurons with a positive PLA signal. (A6) Representative image of PLA signals in neurons (nuclei stained by DAPI) in rat NAc-core using the second set of antibodies. (B) Representative images of immunohistochemistry using D1R antibody (D1R-Ab) or D2R antibody (D2R-Ab) directly conjugated to Alexa-488 or Alexa-568, respectively, in the NAc-shell. Direct confocal FRET analysis was performed, reflected by FRET efficiency (FRET E) and the distance between the dipoles, less than 10 nm (100 Å). (C) A representative close-up of a single MSN cell body from NAc showing D1R-D2R colocalization (left), D1-D2 heteromer FRET efficiency (center) and relative distance between receptors (right). (D,E) Histograms showing FRET E ratios (D) and distance (E) obtained from MSN cell bodies from NAc ( n = 24). Bars are 10 μm.

Article Snippet: Protein homogenates (300 μg /each condition) from rat NAc were incubated with an anti-D2R antibody (Rabbit, Alomone) at 4°C overnight under gentle rotation.

Techniques: Proximity Ligation Assay, Staining, Immunohistochemistry

Journal: Reproductive biomedicine online

Article Title: Social psychogenic stress promotes the development of endometriosis in mouse.

doi: 10.1016/j.rbmo.2016.11.012

Figure Lengend Snippet: Figure 4 – Immmunoreactivity staining of different markers in ectopic lesions in different groups. (A) Representative immunostaining of ADRB2 in endometrium in CONTROL and SHAM mice and in ectopic endometrium in STRESSED and UNSTRESSED mice. ADRB2 and DRD2 immunoreactivity was both seen primarily in glandular epithelial cells and was localized in the cytoplasm. Scale bar = 125 μm. (B) Representative immunostaining of DRD2, VEGF, CD31, CD41, F4/80, PCNA and α-SMA in the ectopic lesions in UNSTRESSED and STRESSED groups. VEGF immunoreactivity was seen primarily in glandular epithelial cells and was localized in the cytoplasm. CD31 immunostainings were seen mostly in vascular endothelial cells. CD41 shows the extent of platelet aggregation and F4/80 immunoreactivity represents the extent of macrophage infiltration. PCNA immunoreactivity was seen both in glandular epithelial cells and stromal cells were localized in the cell nucleus, but the change of immunoreactivity in glandular epithelial cells was more obvious. α-SMA staining were seen mostly in the stromal component of the ectopic lesions. Scale bar = 125 μm. Mice in stressed (STRS) and unstressed (UNSTRS) groups had undergone endometriosis-inducing surgery, while mice the SHAM group underwent non-endometriosis-inducing surgery. Mice in unstressed (UNSTRS), SHAM and control (CTL) groups were not exposed to stress.

Article Snippet: For negative controls, the immunoglobulin G (IgG) from the rabbit serum (Sigma, Darmstadt, Germany) was used instead of primary antibodies against ADRB2, DRD2, VEGF, CD31, CD41, α-SMA and PCNA except for F4/80, for which the IgG was from the rat serum (Boster, Wuhan, China) was used, all used at the same concentration with its corresponding primary antibody.

Techniques: Staining, Immunostaining, Control