Figure 5. Expression of NCS-Rapgef2 is required for cocaine-induced phosphorylation of ERK and upregulation of Egr-1/Zif268 in mouse brain. A, A majority of cocaine-induced phospho-ERK (pERK) activation (10 min after administration of 20 mg/kg cocaine, i.p.) was in D1 receptor-positive neurons in mPFC, NAc, and BLA. In PFC, 56.7 ± 2.8% of pERK+ neurons in layer II/III, 78.1 ± 2.4% of pERK+ neurons in layer V, and 97.3 ± 0.7% pERK+ neurons in layer VI were also tdTomato-positive. In BLA, 72.2 ± 2.8% of pERK+ neurons were D1 receptor-positive; and in shell and core of NAc almost all (98.6 ± 0.1%) pERK+ neurons were D1-MSNs. Right panels (Aa,Ab,Ac) show the images at higher magnification, outlined by white boxes on the left. Scale bar, 200 µm. B, Phospho-ERK elevation observed 10 min after intraperitoneal administration of 20 mg/kg cocaine in Rapgef2cko/cko (flox) mice is attenuated in mPFC and BLA, but not in NAc, of Camk2α-cre+/−::Rapgef2cko/cko (cKO) mice. Scale bar, 200 µm. C, Egr-1/Zif268 elevation observed 1 h after 20 mg/kg cocaine treatment in Rapgef2cko/cko (flox) mice is attenuated in mPFC and BLA, but not in NAc, of Camk2α-cre+/−::Rapgef2cko/cko (cKO) mice. Scale bar, 100 µm. D, Quantification of phospho-ERK and Egr-1/Zif268 in Rapgef2cko/cko (flox) or Camk2α-cre+/−::Rapgef2cko/cko (cKO) mice after saline or cocaine treatment. Relative number of phospho-ERK-positive neurons or Egr-1 IR from mPFC, NAc, and BLA of each animal was normalized by the average value from age-matched saline-treated Rapgef2cko/cko (flox) mice. N = 3-6 animals per group. *p < 0.001 (post hoc Bonferroni test following two-way ANOVA). E, Bilateral ablation of NCS-Rapgef2 in NAc impaired phospho-ERK elevation in response to cocaine. AAV9-hSyn-eGFP (ctrl virus) or AAV9-hSyn-cre-eGFP (cre virus) was injected into the NAc of Rapgef2cko/cko mice. Four weeks later, phospho-ERK activation in both sides of NAc was examined by immunohistochemistry after acute cocaine (20 mg/kg, i.p., 10 min) injection. N = 3 or 4 animals per group. *p < 0.001 (post hoc Bonferroni test following two-way ANOVA). F, Cocaine-induced Egr-1/Zif268 increase in the NAc of Rapgef2cko/cko mice with ctrl virus was impaired in the NAc with cre virus. Four weeks after viral infusion, mice were killed 1 h after saline or cocaine (20 mg/kg) injection. The tissue from NAc was punched out from 0.5 mm coronal sections of mouse brains and protein lysate was used for Western blot with Egr-1/Zif268 antibody. Total protein loading of each sample was first normalized by GAPDH immunoreactivity (IR). Relative Egr-1/Zif268 IR was obtained by comparing to average of that from NAc of saline-treated Rapgef2cko/cko bilaterally injected with AAV9-hSyn-eGFP (ctrl virus). N = 4 or 5 animals per group. *p < 0.001 (post hoc Bonferroni test following two-way ANOVA). Columns and bars represent mean ± sem and scattered points represent individual values. ns, not significant.