Discrete cues paired with naloxone-precipitated withdrawal from acute morphine dependence elicit conditioned withdrawal responses

Abstract

Acute bolus doses of morphine induce a state of acute opioid dependence as measured by naloxone-precipitated withdrawal. Repeated morphine and precipitated withdrawal experience further enhances naloxone-induced withdrawal severity, partly because of direct neuroadaptation to repeated morphine, and partly because of conditioned associations of context and withdrawal experience. To determine whether a discrete tone/light conditioned stimulus could elicit conditioned withdrawal responses in acute dependence, rats trained on a fixed-ratio-15 operant schedule for food reward received morphine (5.6 mg/kg) 4x at daily or weekly intervals, with each morphine injection followed at 4 h by naloxone (1.0 mg/kg) and an operant session. The conditioned stimulus was presented to a Paired group after each naloxone injection. Separate control groups experienced the conditioned stimulus either at a different time of the day or on a different day of the week than naloxone (Unpaired), received naloxone without any conditioned stimulus exposure [Paired-no conditioned stimulus (Paired-NO CS)] or received vehicle instead of naloxone before conditioned stimulus presentation (NaI-Naive). On the test day, all rats received vehicle before conditioned stimulus exposure. The conditioned stimulus alone reliably suppressed responding in Paired groups relative to control conditions with either daily or weekly intervals between conditioning sessions. The administration of morphine 4 h before conditioned stimulus exposure on the test day was not necessary to observe conditioned withdrawal. Thus, conditioned withdrawal is reliably established to discrete cues associated with naloxone-precipitated withdrawal from acute, infrequent (weekly) opioid exposure.

Fig. 1

Operant responding in Paired (Paired-CS, Paired-No CS) and control (Nal-Naive, Unpaired) groups from Experiment 1 as a function of operant interval (Pre-CS, CS 1–10, CS 11–20). A) On Conditioning Days, responding during Pre-CS session (left panel) were suppressed in both the Paired groups as conditioning proceeded (*p < 0.05 vs. Unpaired and Nal-Naive controls), but the effect in the Paired-No CS group was significantly greater than in the Paired-CS group (†p < 0.05, Paired CS vs. Paired-No CS). In the CS 1–10 interval (center panel), responding was predictably suppressed totally within 2 sessions in the Paired groups. A transient decrease in operant responding due to CS novelty followed by habituation was noted in both control groups during the CS 1–10 interval ($p < 0.05, main effect of Conditioning Day). In the CS 11–20 interval (right panel), responding again was predictably suppressed in the Paired groups experiencing naloxone-precipitated withdrawal during operant testing on Conditioning Days. Somewhat unexpected was a modest suppression in responding that emerged over Conditioning Days in both the Nal-Naive and Unpaired control groups ($p< 0.05, main effect of Conditioning Day); this may reflect the emergence of a mild spontaneous withdrawal from repeated daily morphine treatments (see text for further details). B) On Test Day, responding in the Pre-CS session (left panel) was again suppressed in both Paired groups (*p < 0.05 vs. Unpaired and Nal-Naive controls), with the Paired-No CS groups showing greater suppression in this interval than the Paired-CS group (†p < 0.05). Operant responding during the first 10 minutes of CS session (center panel) were suppressed in the Paired-CS group relative to the both control groups(*p < 0.05) and relative to Paired-CS responding in the Pre-CS interval ($p < 0.05 vs. Pre-CS), but responding in the Paired-No CS group actually increased significantly from the Pre-CS to the CS 1-10 interval ($p<0.05 vs. Pre-CS). There was no difference in responding during final 10 minutes of CS session (right panel) between any treatment groups.

Fig. 2

A) Baseline responding across Conditioning and Test weeks for Experiment 2. Operant responding declined in both Paired and Unpaired groups as a function of CS interval (Pre-CS, CS 1–10, CS 11–20). Rats in the Unpaired group showed a modest increase in responding across conditioning weeks, and this increase was not seen in the Paired group (*p < 0.05, treatment × conditioning week interaction). B) Responding on Conditioning Days across weeks. There was no significant change in responding in the Pre-CS interval across Conditioning Weeks. A transient decrease in responding was noted in the CS 1–10 interval when the CS was first introduced to the Unpaired group ($p < 0.05 vs. Pre-CS interval in the corresponding Conditioning Week. This effect was masked in the Paired groups that also received naloxone-precipitated withdrawal (1.0 mg/kg) immediately prior to CS exposure. The decline in responding in the Unpaired group during Conditioning Weeks 1–2 was not present in the CS 11–20 interval; the Paired group showed essentially 100% suppression of responding in the CS 11–20 interval in all Conditioning Weeks.

Fig. 3

Operant responding is suppressed only by cue exposure on the Test Day following 4 weekly Conditioning Sessions in Experiment 2. Operant responding during the Pre-CS interval did not differ between Unpaired and Paired groups. Responding during the CS 1–10 interval were suppressed equally in both Paired groups relative to their corresponding Unpaired control groups (*p < 0.05 vs. Unpaired control), and relative to Pre-CS responding in the same Paired group (†p < 0.05 vs. Pre-CS in same group). Responding during the CS 11–20 interval session were still suppressed in the Paired-Mor group relative to Pre-CS responding in the same group (†p < 0.05), and relative to the Unpaired-Mor group (*p < 0.05 vs. Unpaired control in same CS interval). Both Paired-Mor and Paired-Veh groups showed significant extinction of the conditioned suppression response in the CS 11–20 interval ($p < 0.05 vs. same group in CS 1–10 interval).