Figure 3. Knockdown of OPHN1 prevents the SYN119-mediated removal of synaptic CP-AMPARs in NAc core medium spiny neurons from cocaine-incubated rats.

A, Experimental timeline. Rats underwent 10 days of intravenous (IV) cocaine self-administration (SA) training and a subsequent cocaine-free abstinence period. Rats were infected with scrambled (scr) or Ophn1 shRNA viruses in the first week after SA. On Withdrawal Day 40–60 (WD40–60), rats received an injection of SYN119 (SYN) (10 mg/kg IP) prior to being euthanized for slice recordings.

B, Summary data and representative immunoblots showing expression of OPHN1 protein in the NAc core of drug-naïve rats 4–5 weeks after infection with scrambled (scr) shRNA virus or Ophn1 shRNA virus. All OPHN1 bands were normalized to a GAPDH loading control and then this normalized value was compared between lanes.

C-E, Rats infected with scr or Ophn1 shRNA viruses were injected with SYN 60 min prior to slice recordings to assess the rectification index (RI). SA data for these rats (not shown) were very similar to data presented in Figs. 1 and 2. Shown are representative traces (C) and IV curves (D) of AMPAR-mediated eEPSCs from medium spiny neurons (MSN) recorded at holding potentials from −70 to +40 mV, and the mean RI (E). The scr shRNA group showed a linear IV relationship, indicating that SYN removed CP-AMPARs from synapses. Inward rectification (elevated RI) was observed in the Ophn1 shRNA group, indicating a block of SYN’s effect. Both viruses had a GFP tag that enabled identification of transfected cells. scr 16 cells (6M, 10F)/3 rats (1M, 2F); OPHN1 23 cells (8M, 15F)/6 rats (2M, 4F). For C: Scale bars, 200 pA, 10 ms. For E: Black squares, males; open circles, females.

F-H, Rats were treated exactly as described in C-E except that sensitivity to the CP-AMPAR antagonist NASPM was assessed 60 min after an injection of SYN. Shown in F are example traces of AMPAR eEPSCs measured in SYN-injected rats before (gray; averaged 15 sweeps, −5–0 minutes) and after (blue; averaged 15 sweeps 10–15 minutes) bath application of NASPM (100 μM) (Scale bars, 200 pA, 20 ms). Shown in G is the time course of normalized eEPSCs before and during NASPM application (mean ± SEM). Shown in H is a summary of NASPM effects on eEPSC amplitudes after 10–15 min of NASPM application. NASPM failed to decrease eEPSC amplitude in the scr shRNA group, indicating that SYN had removed CP-AMPARs from synapses, whereas a significant effect of NASPM was found in MSNs expressing Ophn1 shRNA, indicating a block of SYN’s effect by OPHN1 knockdown. scr 6 cells (4M, 2F)/3 rats (1M, 2F); OPHN1 8 cells (3M, 5F)/4 rats (1M, 3F). For H: Black squares, males; open circles, females.

I, AMPA/NMDA ratios for each group. After SYN injection, MSNs expressing Ophn1 shRNA showed a higher AMPA/NMDA ratio (i.e., SYN was not able to remove CP-AMPARs) than MSNs expressing scrambled shRNA (SYN was able to remove CP-AMPARs). scr 16 cells (6M, 10F)/3 rats (1M, 2F); OPHN1 29 cells (10M, 19F)/4 rats (2M, 4F). Black squares, males; open circles, females.

**p<0.01, ***p=0.005, ****p<0.0001, two-sample t-test