Increased Excitability and Synaptic Plasticity of Drd1- and Drd2-Expressing Prelimbic Neurons Projecting to Nucleus Accumbens after Heroin Abstinence Are Reversed by Cue-Induced Relapse and Protein Kinase A Inhibition

Heroin self-administration and relapse in Drd1-Cre⁺ and Drd2-Cre⁺ transgenic LE rats. A, The average number of active lever presses in Drd1-Cre⁺ heroin SA rats (filled red circles, n = 16) was significantly higher than inactive lever presses (filled gray circles), and active lever presses in yoked-saline controls (empty red circles, n = 7; *p < 0.05). B, The average number of active lever presses in Drd2-Cre⁺ heroin SA rats (filled blue circles, n = 11) was significantly higher than inactive lever presses (filled gray circles), and active lever presses in yoked-saline controls (empty blue circles, n = 7; *p < 0.05). C, The average number of heroin infusions received did not differ significantly between Drd1-Cre⁺ and Drd2-Cre⁺ rats. D, Postabstinence cue-induced relapse test: Average number of active lever presses during the first 30 min of the last 3 SA sessions for Drd1-Cre⁺ (red triangles) and Drd2-Cre⁺ (blue triangles) rats (left). Average number of active lever presses during a 30 min cue-induced relapse test for the same rats (right). The number of active lever presses significantly increased for both Drd1-Cre⁺ (**p < 0.01) and Drd2-Cre⁺ (*p < 0.05) rats during the cue-induced relapse test compared with the average number of active lever presses during first 30 min of last 3 SA sessions. Data expressed as mean +/– SEM.

Changes in intrinsic excitability during abstinence from heroin and cue-induced relapse to heroin seeking. A, Schematic of experimental design. After 7 d of home-cage abstinence, a subset of heroin SA rats (n = 8) and all yoked-saline controls (n = 7) were killed for in vitro whole-cell slice electrophysiology. Another subset of heroin SA rats (n = 6) underwent a 30 min cue-induced relapse test immediately followed by in vitro whole-cell slice electrophysiology. B, Representative image of patched D1⁺ PL->NAc PN in bright-field (top) and EGFP fluorescence (bottom). C, Left, The number of APs fired in response to increasing steps of injected current (0–400 pA with 25 pA step increments) in D1⁺ PL->NAc PNs (increased significantly compared with saline controls postabstinence (*p < 0.05) and post-cue-induced relapse test (^p < 0.05, ^^p < 0.01). Right, Representative AP traces at 225 pA injected current (Saline, black; Heroin, red; Heroin + Relapse, orange). D, Representative image of patched D2⁺ PL->NAc PN in bright-field (top) and EGFP fluorescence (bottom). E, Left, The number of APs fired at increasing steps of injected current (0–400 pA with 25 pA step increments) in D2⁺ PL->NAc PNs increased significantly compared with saline controls postabstinence (*p < 0.05, **p < 0.01) and post-cue-induced relapse test (^p < 0.05, ^^p < 0.01). Right, Representative AP traces at 225 pA injected current (saline, black; heroin, blue; heroin plus relapse, purple). F, Left, Rheobase of D1⁺ PL->NAc PNs is decreased significantly during heroin abstinence compared with saline controls (**p < 0.01) and is reversed by cue-induced relapse to heroin seeking. Right, Rheobase in D2⁺ PL->NAc PNs remained unchanged during heroin abstinence and post-cue-induced relapse test. Data expressed as mean +/– SEM.

Abstinence from heroin and cue-induced relapse to heroin seeking changed AP waveform measurements. A, Schematic of rheobase AP trace demonstrating how AP properties were derived. B, Schematic of fAHP amplitude determination. C, Schematic of average trace used to compute mAHP and sAHP amplitude. D, Overlaid representative traces of rheobase measurements used to derive AP waveform measurements. Rheobase trace for Saline (black), Heroin (red), and Heroin-Relapse (orange) in D1⁺ PL->NAc PNs (top) and rheobase trace for Saline (black), Heroin (blue), and Heroin-Relapse (blue) in D2⁺ PL->NAc PNs (bottom). E, F, Changes in AP properties after heroin abstinence and cue-induced relapse. Abstinence from heroin and cue-induced relapse to heroin seeking did not alter AP threshold (E) and amplitude (AP amp) (F). G, AP half-width was not affected by heroin or cue-induced relapse in D1⁺ PL->NAc PNs (left), but it was significantly decreased by heroin (*p < 0.05) and reversed by cue-induced relapse (^p < 0.05) in D2⁺ PL->NAc PNs (right). H, In D1⁺ PL->NAc PNs (left), AP width was not affected significantly by heroin, but cue-induced relapse significantly increased (^p < 0.05) it compared with heroin. In D2⁺ PL->NAc PNs (right), AP width was significantly decreased by heroin (*p < 0.05) and cue-induced relapse normalized it (^^p < 0.01). I, AP depolarization width was unaffected by heroin or cue-induced relapse in D1⁺ PL->NAc PNs (left). AP depolarization width was not affected significantly by heroin in D2⁺ PL->NAc PNs (right) but cue-induced relapse significantly increased (^^p < 0.01). J, AP repolarization width was not affected significantly by heroin in D1⁺ PL->NAc PNs (left) but cue-induced relapse significantly increased it (^^p < 0.01). In D2⁺ PL->NAc PNs (right), AP repolarization width was significantly decreased by heroin (*p < 0.05) and normalized by cue-induced relapse (^^p < 0.01). K, Abstinence from heroin-induced and cue-induced relapse to heroin seeking did not alter fAHP in both D1⁺ and D2⁺ PL->NAc PNs. L, Cue-induced relapse to heroin seeking marginally decreased mAHP in D1⁺ PL->NAc PNs (left) compared with heroin (p = 0.052). In D2⁺ PL->NAc PNs (right), mAHP was marginally decreased after cue-induced relapse to heroin seeking compared with saline controls (p = 0.051), but significantly decreased compared with heroin (**p < 0.01). M, Abstinence from heroin marginally increased sAHP in D2⁺ PL->NAc PNs (right) compared with D1⁺ PL->NAc PNs (left; p = 0.054). However, abstinence from heroin and cue-induced relapse did not affect sAHP in either D1⁺ or D2⁺ PL->NAc PNs. Data expressed as mean +/– SEM.

Effects of in vitro PKA inhibition on intrinsic excitability of D1⁺ and D2⁺ PL->NAc PNs after heroin abstinence. A, Schematic of experimental design. After 6 d of home-cage abstinence, both heroin and yoked-saline SA rats underwent in vitro whole-cell slice electrophysiology with or without in vitro application of Rp-cAMPs. B, Heroin abstinence induced increased firing of D1⁺ PL->NAc PNs (*p < 0.05; left) was reduced to baseline/control levels by in vitro application of RP-cAMPs (^p < 0.05, ^^p < 0.01). The number of APs fired was unaffected by RP-cAMPs application in saline-treated rats. Example traces of AP firing at 225 pA of D1⁺ PL->NAc PNs (Saline, black; Heroin, red; Saline-RP-cAMPs, gray; Heroin-RP-cAMPs, pink; right). C, Heroin abstinence-induced increased firing of D2⁺ PL->NAc PNs (*p < 0.05, **p < 0.01; left) was reduced to baseline/control levels by in vitro application of RP-cAMPs (^p < 0.05, ^^p < 0.01). The number of APs fired was unaffected by RP-cAMPs application in saline-treated rats. Example traces of AP firing at 225 pA of D2⁺ PL->NAc PNs (Saline, black; Heroin, blue; Saline-RP-cAMPs, gray; Heroin-RP-cAMPs, green; right). D, Rheobase of D1⁺ PL->NAc PNs (left) is decreased significantly during heroin abstinence compared with controls (*p < 0.05) but RP-cAMPs treatment did not significantly reverse this effect. Rheobase in D2⁺ PL->NAc PNs (right) remained unchanged during abstinence and was unaffected by in vitro RP-cAMPs application. Data expressed as mean +/– SEM.

Effects of in vitro PKA inhibition on AP waveform measurements of D1⁺ and D2⁺ PL->NAc PNs after heroin abstinence. A, B, Abstinence from heroin or in vitro RP-cAMPs treatment did not affect AP threshold (A) or amplitude (AP amp) (B) of D1⁺ and D2⁺ PL->NAc PNs. C, In D1⁺ PL->NAc PNs (left), heroin abstinence did not affect AP half-width, but RP-cAMPs significantly increased it in heroin-treated rats (^^p < 0.01), while in D2⁺ PL->NAc PNs (right), heroin abstinence significantly decreased AP half-width (*p < 0.05) and RP-cAMPs restored it to baseline levels in heroin-treated rats (^^p < 0.01). D, In D1⁺ PL->NAc PNs (left), heroin abstinence did not significantly decrease AP width, but RP-cAMPs restored it to baseline levels in heroin-treated rats (^p < 0.05). In D2⁺ PL->NAc PNs (right), heroin decreased AP width (**p < 0.01) and RP-cAMPs restored it to baseline levels (^p < 0.05). E, In D1⁺ PL->NAc PNs (left), heroin abstinence did not significantly decrease AP depolarization width, but RP-cAMPs restored it to baseline levels in heroin-treated rats (^^p < 0.01). In D2⁺ PL->NAc PNs (right), heroin significantly decreased AP depolarization width (**p < 0.01) and RP-cAMPs restored it to baseline levels in heroin-treated rats (^^p < 0.01). F, In D1⁺ PL->NAc PNs (left), heroin abstinence did not significantly decrease AP repolarization width, but RP-cAMPs restored it to baseline levels in heroin-treated rats (^^p < 0.01) while in D2⁺ PL->NAc PNs (right), heroin significantly decreased AP depolarization width (**p < 0.01) and RP-cAMPs restored it to baseline levels in heroin-treated rats (^p < 0.05). G, fAHP remained unaffected in D1⁺ PL->NAc PNs (left). In D2⁺ PL->NAc PNs (right), fAHP was significantly reduced in heroin-RP-cAMPs group compared with Saline-RP-cAMPs (*p < 0.05). H, RP-cAMPs treatment significantly reduced mAHP in D1⁺ PL->NAc PNs (left) of heroin-treated rats (*p < 0.05) and saline controls treated with RP-cAMPs (*p < 0.05) but did not affect mAHP in D2⁺ PL->NAc PNs (right). I, sAHP remained unaffected by heroin abstinence and RP-cAMPs treatment in both D1⁺ and D2⁺ PL->NAc PNs. Data expressed as mean +/– SEM.

Effects of heroin abstinence, cue-induced relapse, and in vitro RP-cAMPs treatment on sEPSC amplitude and frequency (freq) of D1⁺ and D2⁺ PL->NAc PNs. A, Abstinence from heroin significantly increased the AP amp of sEPSCs in D1⁺ PL->NAc PNs (left) compared with saline controls (**p < 0.01) and was normalized by cue-induced relapse (^^p < 0.01); sEPSC frequency of D1⁺ PL->NAc PNs (right) was unaffected by heroin abstinence or cue-induced relapse. B, Example traces of sEPSCs of D1⁺ PL->NAc PNs. C, sEPSC amplitude (left) and frequency (right) of D2⁺ PL->NAc PNs remained unaffected by heroin abstinence or cue-induced relapse. D, Example traces of sEPSCs of D2⁺ PL-> PNs. E, Abstinence from heroin significantly increased the amplitude of sEPSCs in D1⁺ PL->NAc PNs (left) compared with saline controls (**p < 0.01) and was normalized by in vitro RP-cAMPs treatment (^^p < 0.01), whereas sEPSC frequency of D1⁺ PL->NAc PNs (right) was unaffected by heroin abstinence and in vitro RP-cAMPs treatment. F, Example traces of sEPSCs of D1⁺ PL->NAc PNs. G, In D2⁺ PL->NAc PNs, both sEPSC amplitude (right) and frequency (right) remained unaffected by heroin abstinence and in vitro RP-cAMPs treatment. H, Example traces of sEPSCs of D2⁺ PL-> PNs. Data expressed as mean +/– SEM.