Addicts repeatedly relapse to drug seeking even after years of abstinence, and this behavior is frequently induced by the recall of memories of the rewarding effects of the drug. Established memories, including those induced by drugs of abuse, can become transiently fragile if reactivated, and during this labile phase, known as reconsolidation, can be persistently disrupted. Here we show that, in rats, a morphine-induced place preference (mCPP) memory is linked to context-dependent withdrawal as disrupting the reconsolidation of the memory leads to a significant reduction of withdrawal evoked in the same context. Moreover, the hippocampus plays a critical role in linking the place preference memory with the context-conditioned withdrawal, as disrupting hippocampal protein synthesis and cAMP-dependent-protein kinase A after the reactivation of mCPP significantly weakens the withdrawal. Hence, targeting memories induced by drugs may represent an important strategy for attenuating context-conditioned withdrawal and therefore subsequent relapse in opiate addicts.

The environmental context of an experience induced by a drug of abuse is a powerful determinant of drug-seeking behavior and relapse in addicts (1, 2), and a wealth of evidence indicates that learning and memory, and particularly contextual memories, play a critical role in establishing conditioned responses to drugs of abuse (3–6). Contextual representations and memories are known to critically recruit the hippocampus, a brain region that plays a major role in processing the learning of associations between the environmental context and unconditioned stimuli (7) (e.g., drugs of abuse). However, the precise role of the hippocampus in the development of drug-induced conditioning and addiction is still unclear. Furthermore, it is unknown how and whether contextual memories of the effect of the drugs are linked to other drug-induced behaviors such as relapse and withdrawal.

A general feature of memory is that, initially, it exists in a labile state and, over time, it undergoes a process of stabilization known as consolidation (8). Established memories can however become again transiently fragile if reactivated for example by retrieval or reexposure to reinforced stimuli (9–12). This phase of postreactivation vulnerability is known as reconsolidation, because during this period the memory returns to a stable state. Reconsolidation, like consolidation, requires new RNA and protein synthesis, as well as the functional role of specific molecular pathways, including those activated by cAMP and cAMP-dependent protein kinase A (PKA) (9–12). Thus, the disruption of these mechanisms following memory reactivation represents a strategy for weakening pathogenic memories including those involved in drug conditioning (12). Morphine- or cocaine-conditioned place preference, as well as the conditioned reinforcing properties of a cocaine-associated stimulus, can be disrupted by postreactivation inhibition of protein synthesis, ERK, or of the expression of the immediate-early gene Zif268 (13–16). We previously showed that, in rats, a conditioned place preference evoked by morphine [m-chlorophenylpiperazine (mCPP)] is persistently disrupted by systemic or stereotactic injections of protein synthesis inhibitors into the hippocampus, amygdala, or nucleus accumbens following its reactivation by a conditioning trial (14).

In this study, we investigated three questions. First, is the contextual memory induced by experiencing a drug of abuse directly linked to motivational withdrawal, a response known to play a major role in relapse and the establishment of addiction? Second, if such a link exists, what is the role of the hippocampus? Finally, is this region involved in establishing a contextual link between the representation of the reinforcing effects of the drug and the withdrawal?

Results

Disrupting the Contextual Memory Evoked by Morphine Weakens Subsequent Withdrawal in the Same Context. We first tested whether disrupting mCPP by inhibiting protein synthesis systemically after its reactivation affects subsequent motivational withdrawal. The protocol consisted of three parts: morphine conditioning (i.e., mCPP), reactivation of mCPP, and induction of withdrawal (Fig. 1A). Rats were conditioned for four consecutive days to 10 mg/kg morphine in a counterbalanced fashion. One week later, mCPP memory was reactivated with a single 10-mg/kg conditioning trial (1XRC), and immediately after, half the animals received two systemic cycloheximide (CXM) injections at 2.2 mg/kg, 5 h apart, and the other half received two injections of vehicle solution. This cycloheximide treatment has been shown to block more than 70% of protein synthesis in the rat brain for at least 6 h and to persistently disrupt mCPP reconsolidation (14). The next day, both groups of rats were divided in two subgroups, which underwent 0.3 mg/kg naltrexone (NTX)–precipitated withdrawal or a control vehicle treatment (see Materials and Methods for details) (17). The withdrawal protocol consisted of NTX administration 4 h after a single morphine conditioning (17). This protocol elicits a significant conditioned place aversion, which is known to be a sensitive and accurate index of the aversive motivational consequences of withdrawal (18). Two days later, all four groups (vehicle/vehicle; CXM/vehicle; vehicle/NTX; CXM/NTX) were tested (test 1) for place preference or place aversion as indices of morphine seeking or morphine withdrawal, respectively. To test whether the effect was persistent, all four groups were retested 1 wk later (test 2). To determine whether place conditioning could be recovered following the reexperience of a new morphine conditioning trial, 24 h after test 2, animals from vehicle/vehicle and CXM/vehicle groups underwent a single conditioning trial and were tested 24 h later (saving; test 3). Finally, to test whether place preference could be recovered in a state-dependent condition, 3 d later, the same animals received an injection of morphine and were then immediately tested (priming injection; test 4). Fig. 1. Disrupting mCPP reconsolidation disrupts subsequent NTX-precipitated withdrawal. Experimental timelines are shown above each experiment. The score values are shown in Table S1. Values of preference or avoidance are expressed in seconds as differences (test vs. pretest) and shown as means ± SEM (A) Cycloheximide significantly disrupts mCPP compared with vehicle (tests 1–4, n = 6–8; *P < 0.05, **P < 0.01). The animals with disrupted CPP also show a significantly disrupted NTX-precipitated withdrawal (tests 1 and 2, n = 7–8; **P < 0.01, ***P < 0.001). (B) Same as in A, except that 1XRC was omitted and testing was completed at test 1; n = 8 per group. A two-way ANOVA comparing preference scores across treatment (vehicle/vehicle, CXM/vehicle) and test (tests 1–4) revealed a significant effect of treatment (F 1,48 = 42.50; P < 0.0001), no effect of test (F 3,48 = 0.30; P = 0.81), and no test–treatment interaction (F 3,48 = 0.17; P = 0.91). A Bonferroni post hoc test revealed that, compared with vehicle, cycloheximide significantly disrupts mCPP at test 1 (P < 0.05; Fig. 1A). The disruption was persistent at test 2 (P < 0.01), test 3 (P < 0.05), and test 4 (P < 0.01), suggesting that the loss of mCPP is persistent and not state-dependent. Notably, the rats that had a disrupted mCPP by cycloheximide and subsequently received NTX also showed a significant loss of withdrawal compared with the group that had an intact mCPP and received NTX, which, as expected, underwent significant withdrawal at test 1 (Fig. 1A). A two-way ANOVA across treatment (CXM/NTX, vehicle/NTX) and test (tests 1 and 2) revealed a significant effect of treatment (F 1,26 = 33.33; P < 0.0001), no effect of test (F 1,26 = 1.54; P = 0.23), and no test–treatment interactions (F 1.26 = 0.55; P = 0.46). Bonferroni post hoc test revealed that, compared with vehicle, cycloheximide disrupted NTX-precipitated avoidance at test 1 (P < 0.001) and the disruption was persistent at test 2 (P < 0.01). Furthermore, the effect on both mCPP and withdrawal was dependent on the reactivation of mCPP. Indeed, rats that underwent the same conditioning and testing schedule described above but received cycloheximide injections in the absence of the reactivating conditioning (Fig. 1B) had normal mCPP (Student t test, P = 0.76) and normal withdrawal (Student t test, P = 0.88). The effect of disrupting mCPP on withdrawal was maintained over time. We performed the same experiment as described earlier, except that the NTX-precipitated withdrawal was elicited 1 wk after the reactivation session followed by cycloheximde or vehicle. As shown in Fig. S1, mCPP was significantly disrupted by cycloheximide (vehicle/vehicle vs. CXM/vehicle; t test, P < 0.001) and this disruption also led to a significant loss of withdrawal (CXM/NTX vs. vehicle/NTX; t test, P < 0.001). Thus, disrupting mCPP with postreactivation injection of protein synthesis inhibitors also significantly weakens withdrawal and the effect is contingent on the reactivation of the memory. This suggests that an associative link must exist between the conditioned contextual representation (i.e., mCPP) and a subsequent withdrawal response.

Drug Conditioning Is Required for Linking Motivational Withdrawal to Memory. If a link exists between an established conditioned place preference and withdrawal and it is the result of repeated contextual conditioning, omitting the conditioning (i.e., no mCPP memory) should produce a normal withdrawal. Furthermore, this withdrawal should not be affected by prior inhibition of protein synthesis following a single conditioning. To test this hypothesis, rats received a single 10-mg/kg morphine conditioning followed by two 2.2-mg/kg cycloheximide injections and, 24 h later, underwent the NTX-precipitated withdrawal protocol. As depicted in Fig. 2A, a single morphine conditioning induced only a small preference at test 1, which was disrupted, although not significantly, by cycloheximide (t test, P = 0.1). Cycloheximide did not affect NTX-induced withdrawal (CXM/NTX vs. vehicle/NTX; t test, P = 0.58, Fig. 2A). Hence, an established conditioning appears to be essential for making a link between mCPP memory and subsequent withdrawal. Furthermore, these data exclude that the effect on withdrawal seen in Fig. 1 results from a direct effect of cycloheximide on the withdrawal. Fig. 2. The contextual conditioning to morphine is necessary for linking a context-precipitated withdrawal. Experimental timelines are shown above each experiment. The score values are shown in Table S1. Preference or avoidance are expressed in seconds as differences (test vs. pretest) and shown as means ± SEM (A) Cycloheximide treatment following a single morphine conditioning (n = 7–8 per group) shows no effect on withdrawal. (B) Four vehicle-context exposures were experienced instead of morphine conditioning. One week later, one morphine conditioning was administered in one context (context A); 24 h later, the withdrawal protocol was carried out in the same context. No effect on withdrawal is found; n = 6–8 per group. (C) Morphine conditioning (4×) 1 wk later: 1XRC was administered following cycloheximide or vehicle treatment. Twenty-four hours later 1× conditioning followed 4 h later by NTX was performed in the vehicle-paired (unpaired) context. Cycloheximide significantly disrupts mCPP (n = 7–8; **P < 0.01), but no effect on withdrawal was found. All together, these results lead to a twofold conclusion. First, the representation of a place memory of the rewarding effects of a drug is linked to and critically influences the representations of other types of drug-induced behaviors such as withdrawal. Second, disrupting the memory, in fact, weakens withdrawal. Given the fact that the withdrawal is linked to the contextual memory, we then asked the question: is a repeated preexposure to a context (without drug pairing) sufficient to create a contextual representation that becomes linked to withdrawal when precipitated by NTX in the same context? Or is the contextual conditioning to the drug necessary? As depicted in Fig. 2B, rats underwent the same experimental protocol as described in Fig. 1A except that they received a 4-d exposure to a conditioning box but were injected with vehicle and not morphine. A week later, they received one morphine conditioning in the same context followed by cycloheximide or vehicle treatment, and the following day were exposed to the withdrawal (one morphine conditioning followed by NTX and context exposure) or relative control protocol (one morphine conditioning followed by vehicle and context exposure), as detailed in Materials and Methods. Two morphine exposures induced only a small preference which was disrupted, although not significantly, by cycloheximide (t test, P = 0.08). Furthermore, cycloheximide did not affect NTX-precipitated withdrawal, as both cycloheximide and vehicle-injected groups showed a strong and comparable aversion (t test, P = 0.2; Fig. 2B). Hence, the withdrawal linked to the contextual memory is established as a consequence of the place conditioning to morphine. Given these results, we then asked whether disrupting the mCPP memory only affects withdrawal evoked in the conditioned context. We repeated the experiment described earlier in Fig. 1A, but carried out the withdrawal protocol in the vehicle-paired compartment. Specifically, rats received 4-d morphine conditioning and, 1 wk later, the 1XRC followed by cycloheximide or vehicle injections. Twenty-four hours later, they underwent the NTX-precipitated withdrawal protocol or vehicle-control treatment in the context that during conditioning was paired with vehicle. Therefore, rats experienced this context for the same duration and frequency as that paired with morphine, but this context was never paired with the drug during the 4 d of conditioning; thus, the rats experienced the drug only once during the withdrawal protocol. Student t test revealed that, compared with vehicle, cycloheximide significantly disrupted mCPP (P < 0.01; Fig. 2C), as expected. However, the rats that received cycloheximide showed a NTX-precipitated withdrawal (CXM/NTX) in the vehicle-paired context comparable to that of vehicle-treated controls (vehicle/NTX). These data show that the withdrawal that is precipitated in the morphine-conditioned environment has also become conditioned to this context and it is different from the withdrawal precipitated by NTX in a new context that was not previously conditioned to the drug. Hence, our findings indicate that, following contextual conditioning to a drug of abuse, the drug-paired contextual representation is a critical component of the withdrawal elicited in the same context. When the memory of the drug-associated contextual experience is disrupted, the subject fails to show withdrawal in the same context. Finally, we investigated whether the effect of mCPP memory disruption on withdrawal impact physical signs. Rats received morphine conditioning and, 1 wk later, mCPP reactivation followed by cycloheximide or vehicle treatments as described earlier for the experiment in Fig. 1A. NTX-precipitated withdrawal was induced 24 h later by one morphine conditioning followed, 4 h later, by 3 mg/kg of NTX s.c. Immediately after, the rats were confined to the morphine-conditioned compartment and videotaped for 15 min to score their physical signs of withdrawal (19). As shown in Table S2, no difference in global rating or severity of individual somatic signs was seen in cycloheximide versus vehicle-injected groups. Thus, postreactivation disruption of mCPP, which leads to significant weakening of context-dependent withdrawal, targets motivational but not physical withdrawal.