Conditioned Effects of Heroin on the Expression of Inducible Nitric Oxide Synthase in the Rat Are Susceptible to Extinction and Latent Inhibition
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Psychopharmacology (2007) 191:879–889 DOI 10.1007/s00213-006-0673-z ORIGINAL INVESTIGATION Conditioned effects of heroin on the expression of inducible nitric oxide synthase in the rat are susceptible to extinction and latent inhibition Jennifer L. Szczytkowski & Donald T. Lysle Received: 24 March 2006 /Accepted: 9 December 2006 / Published online: 9 January 2007 # Springer-Verlag 2007 Abstract Results The results showed that both extinction and latent Rationale The administration of heroin has been shown to inhibition reduced the conditioned effects of heroin on the inhibit the induction of nitric oxide, a molecule known to production of nitric oxide. play a critical role in immune function. Previous research Conclusion This study provides the first evidence that the has shown that this alteration can be conditioned to conditioned effects of heroin on nitric oxide production environmental stimuli that have been associated with drug follow accepted principles of learning. administration. However, it remains unknown whether the conditioned effects of heroin on nitric oxide formation Keywords Nitric oxide . Heroin . Diacetylmorphine . follow accepted principles of learning. Lipopolysaccharide . Conditioning . Extinction . Objective This study sought to determine whether manip- Latent Inhibition ulations that induce extinction and latent inhibition, two learning paradigms known to reduce the expression of conditioned responses, would alter heroin’s conditioned Introduction effects on the expression of inducible nitric oxide synthase (iNOS). The high prevalence of opportunistic infections among Materials and methods The conditioning procedure in- heroin users has long suggested that heroin use may alter volved repeated pairing of heroin administration with resistance to infectious disease (Luttgens 1949; Hussey and placement into a standard conditioning chamber. Rats were Katz 1950; Louria et al. 1967;Gartenetal.2004). repeatedly exposed to the chambers without heroin rein- Although there has been speculation that this high rate of forcement to determine whether the conditioned response infection may be due to non-sterile intravenous administra- would extinguish. To induce latent inhibition, rats received tion of drug, there is increasing evidence suggesting that repeated exposure to the chamber before the start of opiates directly alter immune status (Brown et al. 1974; conditioning to inhibit the acquisition of the conditioned McDonough et al. 1980; Nair et al. 1986; Donahoe et al. response. Ten days after the final conditioning session, all 1986; Novick et al. 1989; Oschorn et al. 1990; Govitrapong rats were injected with lipopolysaccharide (LPS) to induce et al. 1998; Sharp et al. 2001). Studies in our laboratory iNOS expression. Spleen and liver tissue were removed to have shown that heroin induces alterations in a number of determine iNOS expression using reverse transcriptase immunological parameters including alterations in natural polymerase chain reaction. Blood was collected to deter- killer cell activity, T- and B-lymphocyte proliferation, and mine the concentration of nitrite/nitrate. nitric oxide production (Fecho et al. 2000; Lysle and How 2000). The reduction in nitric oxide production induced by : heroin administration is dose-dependent and appears to be J. L. Szczytkowski D. T. Lysle (*) mediated through opioid receptors, as these alterations are Department of Psychology and Curriculum in Neurobiology, reversed upon administration of the opioid antagonist, CB#3270, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3270, USA naltrexone (Lysle and How 2000). The production of nitric e-mail: [email protected] oxide by immune cells, particularly macrophages, provides 880 Psychopharmacology (2007) 191:879–889 a substantial degree of microbial resistance (Green et al. may produce a weakening or inhibition of this response. 1990; Nathan and Hibbs 1991; Vincendeau et al. 1992; Two of the most widely studied experimental paradigms Rossi et al. 1999). Mice lacking the gene for inducible leading to reduced expression of the CR are extinction and nitric oxide synthase show an increased susceptibility to latent inhibition. Extinction is evident when after condi- parasitic and bacterial infections (MacMicking et al. 1995; tioning has taken place, repeated exposure to the CS Weietal.1995; Lindgren et al. 2004). The production of without the US decreases the CR. Latent inhibition is a large quantities of nitric oxide by macrophages also process by which repeated non-reinforced exposure to a provides resistance to viral infections and displays tumor stimulus before conditioning will inhibit the formation of cytotoxicity (Karupiah et al. 1993; Chang et al. 2003; a CR to that stimulus (Lubow and Moore 1959). Extinction Hrabak et al. 2006). In addition to its role as an anti- and latent inhibition have been studied extensively within microbial agent, nitric oxide also serves many immunoreg- models of conditioning to test hypotheses concerning ulatory functions. There is evidence that nitric oxide retroactive and proactive stimulus interference, respectively suppresses the formation of antibodies to tetanus toxoid (Pineno and Miller 2005). The susceptibility of condi- and sheep red blood cells after Salmonella typhimurium tioned, heroin-induced immune alterations to the effects of immunization (Al-Ramadi et al. 1992; Eisenstein et al. extinction and latent inhibition demonstrate that this 1994). Numerous studies have also shown an anti-prolifer- conditioning paradigm is a true form of associative learning ative effect of nitric oxide on lymphocytes (Albina and and adheres to accepted principles of learning. Furthermore, Henry 1991). Thus, nitric oxide plays a critical role in given the important health consequences that may result immune processes and may be involved in the altered from conditioned immune alterations, the assessment will susceptibility to infection evident in heroin users. identify procedures that influence the CR. Although it is now apparent that heroin impairs immune Given this information, it was hypothesized that both status, there has been limited research on how conditioned pre- and post-exposure to the drug-paired environment (i.e., drug cues may also induce changes in immune function. the CS) should reduce the conditioned effects of heroin on Classical or Pavlovian conditioning is a well-characterized nitric oxide production. To test this hypothesis, the present learning phenomenon in which a formerly neutral stimulus study evaluated the effects of two behavioral manipula- [the conditioned stimulus (CS), such the conditioning tions, pre- and post-exposure to the CS, on the conditioned chamber] will begin to elicit a response [the conditioned suppression of inducible nitric oxide synthase (iNOS). response (CR)] after repeated pairing with a biologically Previous research in our laboratory has shown that heroin relevant stimulus [the unconditioned stimulus (US), such as induces a dose-dependent reduction in lipopolysaccharide heroin] that had elicited a response upon its first presenta- (LPS)-induced iNOS mRNA expression in the spleen and tion (Pavlov 1927). Several investigators have shown that liver. The findings reported here are important because they many of the physiological and behavioral responses to indicate that previously heroin-associated environmental drugs of abuse may be conditioned to previously drug- stimuli are not only capable of inducing alterations in nitric paired stimuli. For example, environmental stimuli that had oxide expression but that these effects may be modified by previously been paired with morphine administration can manipulations of the relationship between the environment elicit such morphine-like effects as hyperthermia when and drug delivery. These results further implicate a role for presented in the absence of morphine (Miksic et al. 1975; associative learning processes in the conditioned effects of Eikelboom and Stewart 1979; Schwarz and Cunningham heroin on nitric oxide and suggest that these effects may be 1990). In line with these studies, our laboratory has recently mediated, at least in part, by centrally located neural provided new data indicating that heroin’s effects on nitric substrates of learning. oxide expression may be conditioned to environmental stimuli. In those investigations, rats received subcutaneous injections of heroin (1 mg/kg) upon placement into a Materials and methods distinctive environment which served as the CS. When rats were subsequently re-exposed to the environment without Animals further heroin administration, the production of nitric oxide was suppressed similar to that seen with heroin adminis- Male Lewis rats, weighing 225–250 g, were purchased tration alone (Lysle and Ijames 2002). These data provided from Charles River Laboratories (Raleigh, NC, USA). the first evidence that heroin-induced alterations in nitric Upon arrival, animals were housed individually in plastic oxide expression may be conditioned to environmental cages in a colony room with a reversed light–dark (12 h) stimuli. cycle maintained through artificial illumination. The ani- The development and persistence of the CR is dependent mals were allowed access to food and water ad libitum on several factors, and certain experimental manipulations throughout the experiment. All animals were given a 2- Psychopharmacology (2007) 191:879–889 881 week habituation period before the start of experimental Extinction manipulations and were handled regularly