Inactivation of NMDAR and CaMKII signaling within the prelimbic cortex blocks incubated cocaine- and sucrose-craving

Abstract

The incubation of craving is a term coined to characterize the behavioral phenomenon wherein cue-elicited craving strengthens over a period of abstinence. Incubated cocaine-craving is mediated, at least in part, by increased glutamate release within the prelimbic cortex (PL). We hypothesized that this glutamate release stimulates NMDA-type glutamate receptors (NMDARs) leading to calcium-dependent activation of CaMKII signaling that drives incubated craving. To test this hypothesis, adult male and female Sprague-Dawley rats were trained to self-administer either IV cocaine or sucrose pellets (6h/day x10 days) and tested for cue-elicited cocaine- or sucrose-craving in early versus later (i.e. after incubation) withdrawal. Incubated cocaine-seeking was associated with increased CaMKII activity in the PL, but no change in NMDAR subunits. In contrast, incubated sucrose-craving was associated with many sex-dependent changes in both NMDAR subunit expression and CaMKII activation that were subregion-selective. An intra-PL infusion of the NMDA antagonist D-AP5 (2.5 or 7.5 μg/side) or the CaMKII inhibitor myr-AIP (10 pg/side) blocked both incubated cocaine- and sucrose-craving, with no effects detected in early withdrawal. Co-infusion of both D-AP5 and myr-AIP exerted an additive effect on incubated cocaine-craving that was larger than either antagonist alone. These data corroborate earlier evidence for distinct biochemical correlates within mPFC between incubated cocaine- and sucrose-craving and, for the first time, demonstrate that NMDARs and CaMKII activation within the PL are common drivers of incubated craving that operate via independent signaling pathways suggesting combined pharmacological treatments may have greater efficacy in managing addiction.

Keywords: NMDA receptor; calcium/calmodulin-dependent kinase; incubation of craving; prefrontal cortex; sex differences.

Figure 1:. Immunoblotting conducted on the PL subregion of cocaine-incubated rats.

(A) Active lever-pressing exhibited by male and female rats tested for incubated cocaine-craving. Details of the statistical analyses are provided in Ref. . Immunoblotting for the total protein expression of (B) GluN1 [F(1,44)<0.026, p>0.873], (C) GluN2A [F(1,44)<0.020, p>0.888] and (D) GluN2B [F(1,44)<0.002, p>0.968] within the PL of male rats indicated no differences between cocaine-experienced rats (Coc) and cocaine-naive controls (Control), when examined on withdrawal days 3 or 30 (respectively, WD3 and WD30). Comparable immunoblotting conducted on the PL of females also indicated no group differences in (E) GluN1 [F(1,47)<3.731, p>0.060] or (F) GluN2A [F(1,47)<2.424, p>0.127], while a Withdrawal effect was detected for GluN2B (G) [F(1,47)=1.380, p=0.05]. Immunoblotting for the total protein expression of (H) CaMKII, (I) p(Thr286)-CaMKII and (J) their relative expression within the PL subregion of male rats. In contrast to Controls, cocaine-experienced males exhibited a time-dependent increase in both total [for Controls: t(1,17)=0.503,p=0.622; for Cocaine: t(1,18)=2.119, p=0.048] and relative expression of p(Thr286)-CaMKII [for Control: t(1,17)=−0.373, p=0.714; for Cocaine: t(1,19)=−4.017, p<0.001]. Comparable immunoblotting conducted on PL tissue from females (K-M) revealed a similar pattern of group differences for both total [for Controls: t(1,17)=0.265, p=0.794; for Cocaine: t(1,23)=−2.623, p=0.015] and relative expression of p(Thr286)-CaMKII [for Control: t(1,17)=0.454, p=0.656; for Cocaine: t(1,22)=2.508, p=0.020]. Representative immunoblots are provided for all proteins examined. The data represent the means ± SEMs of the individual animals indicated. *p<0.05 vs. Control (main Drug effect); +p<0.05 vs. WD3 (Withdrawal effect).

Figure 2:. Effects of an intra-PL infusion of D-AP5 on cue-elicited cocaine- and sucrose-seeking during protracted withdrawal.

Summary of the effects of microinjection with vehicle (VEH), 2.5 μg or 7.5 μg/side D-AP5 on the number of active (left) and inactive (right) lever-presses emitted during an initial test for cue-elicited cocaine-craving conducted in later withdrawal (WD30+). (A) Relative to early withdrawal controls (WD1-VEH), WD30+-VEH males and WD30+−2.5 AP5 males exhibited incubated craving [for VEH: t(26)=4.435, p<0.001; for 2.5 μg D-AP5: t(36)=3.912, p<0.001], the magnitude of which was reduced by the higher D-AP5 dose [t(53)=4.321, p<0.001], down to the level of WD1 controls [t(29)=1.942, p=0.062]. When carry-over effects were examined the following day (WD31+) (C), the attenuating effect of 7.5 μg/side D-AP5 on active lever-pressing was no longer present [t(48)=.046, p=0.964], and all three groups exhibited incubated responding versus WD1-VEH controls [for VEH: t(48)=6.999, p<0.001; for 2.5 μg D-AP5: t(35)=5.355, p<0.001; for 7.5 μg D-AP5: t(42)=7.925, p<0.001]. (B,D) Relative to WD1-VEH controls, rats infused with either VEH or 7.5 μg/side D-AP5 exhibited higher inactive lever-pressing on both cue test days than WD1-VEH controls [Test 1, for VEH: t(51)=4.101, p<0.001; for 7.5 μg AP5: t(41)=4.221, p<0.001; Test 2, VEH: t(53)=3.880, p<0.001; 7.5 μg AP5: t(39)=4.126, p<0.001]. Summary of the results from a comparable study of incubated sucrose-seeking, comparing the effects of VEH versus the 7.5μg/side dose of D-AP5 during protracted withdrawal (WD30). (E) WD30-VEH controls expressed incubated sucrose-craving [t(27)=4.889, p<0.001], the magnitude of which was reduced by 7.5 μg D-AP5 [t(12)=5.158, p<0.001] to the level of rats tested in early withdrawal [t(22)=1.324, p=0.199], but no group differences were detected for inactive lever-responding during the initial test (F). When carry-over effects were examined (WD31), incubated craving was not longer apparent in VEH-infused controls [t(22)=1.324, p=.12], while rats infused with 7.5 μg D-AP5 exhibited higher active (G) [t(24)=3.033, p<.001] and inactive (H) [t(23)=2.852, p<0.001] lever-presses vs. WD1-VEH controls. Cartoons depicting the placements of the microinjectors within the PL in both experiments are provided. Data represents the means± SEMs of the number of individual rats indicated. *p<0.05 vs. VEH-WD30; +p<0.05 vs. WD1 (withdrawal day 1).

Figure 3:. Effects of an intra-PL infusion of D-AP5, myr-AIP, and their combination on cue-elicited cocaine- & sucrose-seeking during protracted withdrawal.

Summary of the immediate effects of microinjection with vehicle (VEH), 7.5μg/side D-AP5, 10 pg/side myr-AIP or a combination of the two inhibitors on the number of active (left) and inactive (right) lever-presses emitted during an initial test for cue-elicited craving conducted in early (WD1) or later withdrawal (WD30+ or WD40), as well as a test for carry-over effects conducted the following day. (A) WD30+-VEH rats exhibited incubated cocaine-craving [t(71)=6.189, p<0.0001], that was reduced by intra-PL infusion of D-AP5 [t(71)=5.186, p<0.001], myr-AIP [t(71)=4.298, p=0.0005] and their combination [t(71)=6.447, p<0.0001], with all 3 pretreatments reducing active lever-responding to the level of WD1-VEH controls [for D-AP5: t(29)=1.942, p=0.062; for myr-AIP: t(21)=1.851, p=0.078; for combination: t(24)=0.016, p=0.988]. Inhibitor co-infusion also reduced incubated responding to a greater extent than either AP5 [t(71)=2.907, p=0.0065] or myr-AIP alone [t(71)=3.231, p=0.0034], but did not impact inactive lever-pressing (t-tests, p>0.05) (B). (C) When tested the next day, incubated cocaine-craving was apparent in all WD30+ rats, with the exception of the rats with prior co-infusion [WD30+-VEH: t(66)=2.954, p=0.0151; D-AP5: t(66)=3.647, p=.0019; myr-AIP: t(66)=2.794, p=0.0232; co-inhibitor: t(66)=1.759, p=0.2337]. (D) Rats pretreated with myr-AIP emitted fewer inactive lever-presses, relative to both WD30+-VEH controls [t(67)=2.918, p=0.0469] and rats with prior D-AP5 pretreatment [t(67)=3.109, p=0.0272]. In a follow-up sucrose study, VEH-infused rats exhibited incubated sucrose-craving on WD40 [t(18)=3.128, p=0.0058], while that myr-AIP-infused rats was not statistically different from either VEH control (E) [vs. WD40-VEH: t(16)=1.944, p=0.0697; vs. WD1-VEH t(14)=.8942, p=0.3863]. (F) No group differences were apparent for inactive lever pressing on the first Cue Test. When tested the next day, no group differences were detected for active lever-pressing (G), while prior myr-AIP infusion increased inactive lever-pressing relative to both VEH groups (H) [vs. WD1-VEH: t(13)=3.566, p=0.0034; vs. WD41-VEH: t(16)=2.948, p=0.0095]. Cartoons depicting the placements of the microinjectors within the PL in both experiments are provided. Data represents the means± SEMs of the number of individual rats indicated. +p<0.05 vs. WD1, *p<0.05 vs. WD30+VEH, ** p<0.05 vs. D-AP5, *** p<0.05 vs. myr-AIP

Figure 4:. Effects of an intra-PL infusion of D-AP5 or myr-AIP on cue-elicited cocaine-seeking in early withdrawal.

Summary of the effects of an intra-PL infusion of vehicle (VEH), D-AP5 (7.5μg/side) (top) and myr-AIP (10 pg/side) (bottom) on active (left) and inactive (right) lever pressing on a test for cue-elicited cocaine-seeking conducted on withdrawal day 1 (WD1). Both D-AP5 and myr-AIPincreased both active (A) [for D-AP5: t(19)=2.113, p=0.048; for myr-AIP: t(17)=2.813, p=0.0119] and inactive lever-pressing (B) [D-AP5: t(16)=2.587, p=0.019; myr-AIP: t(16)=2.587, p=0.0199]. Cartoons depicting the placements of the microinjectors within the PL in both experiments are provided. The data represent the means ± SEMs of the number of individual rats indicated. *p<0.05 vs. VEH.

Figure 5:. Immunoblotting conducted on the PL subregion of sucrose-incubated rats.

(A) Active lever-pressing of male and female rats tested for incubated sucrose-craving prior to immunoblotting. The data represent the means ± SEMs of the number of rats indicated and details of the statistical analyses are provided in Ref. . Immunoblotting indicated: (B) a Sex and Withdrawal effect for GluN1; (C) a Sex × Withdrawal interaction for GluN2A that reflected a time-dependent decrease in GluN2A in female [t(22)=4.334, p<0.001], but not male [(22)=1.722, p=0.099], rats; (D) a Sex effect and a Sex × Withdrawal interaction for GluN2B, reflecting a time-dependent decrease in GluN2B within the PL of male [t(23)=2.034, p=0.054], but not female rats[t(22)=0.787, p=0.440]; (E,F) Sex effects for both CaMKII and p(Thr286)-CaMKII; and (G) a Sex × Withdrawal interaction for relative p(Thr286)-CaMKII expression that reflected a time-dependent decrease in female [t(19)=3.792, p=0.001], but not male [t(23)=1.529, p=0.140] rats. Representative immunoblots are provided for all proteins examined. The data represent the means ± SEMs of the individual animals indicated. *p<0.05 vs. Control (main Drug effect); +p<0.05 vs. WD3 (Withdrawal effect).