A locus and mechanism of action for associative morphine tolerance

Abstract

Repeated administration of an opioid in the presence of specific environmental cues can induce tolerance specific to that setting (associative tolerance). Prolonged or repeated administration of an opioid without consistent contextual pairing yields non-associative tolerance. Here we demonstrate that cholecystokinin acting at the cholecystokinin-B receptor is required for associative but not non-associative morphine tolerance. Morphine given in the morphine-associated context increased Fos-like immunoreactivity in the lateral amygdala and hippocampal area CA1. Microinjection of the cholecystokinin B antagonist L-365,260 into the amygdala blocked associative tolerance. These results indicate that cholecystokinin acting in the amygdala is necessary for associative tolerance to morphine's analgesic effect.

Fig. 1

Associative tolerance is reversed by CCK-B receptor antagonism. (a) Acquisition of associative tolerance (n = 24) with administration of saline on even-numbered days and morphine (2.5 mg per kg) on odd-numbered days in their associated environments. (b) Associative tolerance is context dependent. Tail-flick latency (n = 19) in morphine-paired and saline-paired environments before (Baseline) and after (Morphine) the acquisition of associative tolerance. Baseline tail-flick latencies were measured on experimental days 1 and 2, respectively. Animals were tolerant to morphine when tested in the morphine-paired environment by the sixth administration (day 13) but showed no evidence of analgesic tolerance to the same dose of morphine given in the saline-paired environment (**p < 0.01). (c, d) Associative tolerance is reversed by the systemic administration of the CCK-B antagonist L-365,260 (0.2 mg per kg, n = 11, p < 0.01) but not by the CCK-A antagonist MK-329 (0.1 mg per kg, n = 13). (e, f) Non-associative tolerance is not reversed by the systemic administration of L-365,260 (0.2 mg per kg, n = 8) or MK-329 (0.1 mg per kg, n = 4). Following the full development of non-associative tolerance, L-365,260 and MK-329 were administered 7 min before systemic morphine. (g–j) Neither pre- nor post-treatment with CCK antagonists reversed partial non-associative tolerance. To avoid a ceiling effect, two groups of animals were implanted with subcutaneous morphine pellets every day for three instead of five days. This produced partial analgesic tolerance, which was unaffected by either L-365,260 (0.2 mg per kg, n = 13) or MK-329 (0.1 mg per kg, n = 13). (g, h) L-365,260 given 45 min after an additional challenge dose of morphine (2.5 mg per kg, i.v., n = 6, gray bar) does not potentiate morphine in partially tolerant rats (black bar, g). As a fall in morphine concentration could account for this failure, we controlled for this possibility by administering L-365,260 7 min before systemic morphine administration (2.5 mg per kg, i.v., n = 7; h). No potentiation was observed (black bar in h not significantly different from gray bar in g). (i, j) MK-329 had no significant effect on analgesia in partially tolerant rats, whether administered 45 min after (i; n = 6) or 7 min before (j; n = 7) a challenge dose of morphine (2.5 mg per kg, i.v.).

Fig. 2

Mean number of Fos-positive cells per section (50 μm) per brain region for associatively and non-associatively tolerant animals (n = 4 animals per group). Increases in Fos expression are observed in amygdala and hippocampus during expression of associative morphine tolerance. NAcc, nucleus accumbens; VTA, ventral tegmental area; CAl, hippocampal area CAl; CeA, central amygdala; BLA, basolateral amygdala; LA, lateral amygdala. (*p < 0.05; **p < 0.01).

Fig. 3

Photomicrographs of Fos-positive cells in the amygdala of associatively and non-associatively tolerant animals. AS, associatively tolerant animals administered saline in the saline-paired environment on experimental day 15; AM, associatively tolerant animals administered morphine in the morphine-paired environment on experimental day 15; NAS, non-associatively tolerant animals administered saline on experimental day 6; NAM, non-associatively tolerant animals administered morphine on experimental day 6.

Fig. 4

Microinjection of the CCK-B receptor antagonist L-365,260 into CNS sites. Abbreviations as in Fig. 2. (a) Comparison of tail-flick latencies following systemic administration (SYST; n = 11) or microinjections of L-365,260 into the L/BLA (n = 12), CeA (n = 7), VTA (n = 7), NAcc core (n = 10) or CA1 (n = 12). White bars, baseline in the morphine-paired context on experimental day 1 or 2; black bars, L-365,260 administered 15 min before morphine on experimental day 15 in the morphine-paired context (**p < 0.0l, *p ± 0.05). (b) Comparison of tail-flick latencies following the administration of L-365,260 or vehicle in either the morphine-paired or saline paired-context. White bars, baseline in the saline-paired and morphine-paired contexts; black bars, L-365,260 administered either 15 min before morphine in the morphine-paired context on experimental day 15 or 15 min before saline in the saline-paired context on experimental day 17; gray bar, vehicle (90% ethanol) administered 15 min before saline in the saline-paired context on experimental day 17.