| Objective:Addiction to drugs is based on pathological changes in brain function produced by repeated pharmacological insult to the brain circuits that regulate how a person interprets and behaviorally responds to motivationally relevant stimuli. Our labs previous findings suggests that cholinergic transmission in?uences heroin self-administration and reinstatement. Therefore, we adopted huperzine A, a Lycopodium alkaloid isolated from Chinese herb Huperzia serrata, is a potent,selective, reversible, and well-tolerated inhibitor of acetylcholinesterase in the present study, focused to observing the effects of huperzine A on heroin reward and reinstatement on heroin seeeking, we also assessed the effect of administration of huperzine A into the laterodorsal tegmental nucleus(LDTg)/pedunculopontine tegmental nucleus(PPTg ) on heroin-seeking triggered by heroin-paired cues. The present data indicate a novel role for the PPTg/LDT in the circuitry mediating heroin cue-induced reinstatement of drug seeking, and represent a potentially useful strategy for the development of a pharmacological treatment for heroin addiction.Methods:Rats were trained to nose poke on a fixed ratio ( FR )5 schedule of food reinforcement (45 mg pellets; Noyes, Lancaster, NH) in operant conditioning chambers ( 30 x 20 x 24 cm high ) during 7 days food training session. The chambers were equipped with two retractable nose pokes, a stimulus light above each nose poke, a food pellet dispenser between the nose pokes, a house light on the wall opposite to the nose pokes. During the session, each nose poke on the active nose poke resulted in delivery of a food pellet only. Nose pokes on the inactive level had no programmed consequence. The effects of acute systemic administration of the huperzine A was examined on sucrose self-administration were measured. The variable include active nose poke responses, in active nose poke responses, the number of sucrose pellets during each training session were recorded by computer.Rats were trained to self-administer heroin under fixed ratio schedule. During each daily 4 h self-administration session, the rats were moved from their home cages to the operant chambers and their connectors were attached to the infusion lines. Each session started with the illumination of the green light inside the active nose-poke hole. The rats received a single heroin infusion ( 50μg/kg per infusion) following completion of the ratio requirement in the active nose-poke. The poke light was turned off during heroin infusions. A 20 s inter-trial interval (time out) followed and then another trial began. Responding in the in active nose-poke had no consequences. The sessions ended after 4 h, whichever occurred first. A progressive ratio schedule PR3-4 was used to evaluate the relative motivational value by measuring the"breaking point". After the heroin training for 14 days, the rats were divided randomly into the groups. The effects of huperzine A on heroin self-administration were observed in the experiment. The variable including the active nose poke responses, inactive nose poke responses were recorded during the trial.Rats underwent heroin self-administration for 14 days, Daily 2-h extinction sessions were conducted until responding was less than 10% of the responses maintained by heroin self administration. This initial extinction criterion was typically met in 10 days. After reaching of extinction criterion, all rats were tested for reinstatement of the drug seeking in the original self-administration context and each animal was tested only once. On the 3th day of withdrawal, rats were challenged by a single dose of heroin ( 0.25 mg/kg ) administration, and active nose poke responses, inactive nose poke responses within 2 hour were recorded. The activity of locomotor within 3 hour was recorded after 14 days of heroin withdrawal.Following 14 days of heroin self-administration and 14 days of extinction as described above, rats were placed in a stereotaxic apparatus and guide cannulae for microinjections were implanted bilaterally, LDTg/ PPTg . Rats were placed into the self-administration chambers 15 min after LDTg/ PPTg huperzine A (1μg/side, 0.5μl), huperzine A (3μg/side, 0.5μl), tetrodotoxin (TTX; 0.3 ng/side, 0.5μl) or vehicle (aCSF 0.5μl). And active nose poke responses, inactive nose poke responses within 2 hour were recorded.Results:1. Effects of huperzine A on sucrose self-administration In order to assess the reinforcer specificity of this huperzine A effect, we assessed the effect of systemic huperzine A ( 30, 100, 300μg/kg ) on the sucrose seeking. These data were analyzed with a one-way analysis of variance, which indicated a lack of a significant treatment effect(F(3.38)=1.179,P=0.331).2. Effects of huperzine A on heroin self-administrationAdministration of huperzine A prior to heroin self-administration under an FR1 schedule of reinforcement dose-dependently decreased heroin intake. A one-way ANOVA on the effect of dose revealed a significant effect ( F (4,70) = 5.118, P = 0.001 ). Post hoc comparisons revealed that the number of infusions self-administered after the 300μg/kg doses of huperzine A were significantly different from baseline conditions ( P<0.001 ). No significant effects were revealedfor responding on the in active poke. The effect of huperzine A on heroin-reinforced responding under a PR schedule of reinforcement dose-dependently decreased heroin intake. The total active nose-poke responses were analyzed with a one-way analysis of variance (ANOVA), which revealed a significant main effect of treatment ( F(3,40) = 5.302, P=0.004 ). Post Hoc Tests showed that the total active responses were significantly different between the huperzine A ( 30,100 and 300μg/kg ) and the vehicle treatments. In contrast, a one-way ANOVA indicated no significant treatment effect in terms of inactive nose-poke responses. Huperzine A produced a dose-dependent decrease in break points ( F(3, 40 )= 4.604, P = 0.007 ) . Post Hoc Tests revealed that the highest doses of huperzine A ( 300μg/kg ) caused significant reductions in responding when compared with vehicle.3. Effect of huperzine A treatment on heroin seeking induced by cues or heroin priming Huperzine A at doses ranging from 50μg/kg to 200μg/kg blocked the increase in responding over baseline extinction( F(3,28) = 10.069, P< 0.0001 ). When compared to responding after vehicle, post-hoc analyses for vehicle vs. Huperzine A showed significant differences at both the 50μg/kg,100μg/kg and 200μg/kg doses ( P<0.01 ). Drug seeking induced by the conditioned cues in the huperzine A pretreatment group was significantly lower than that of the saline group( F(3,30) = 24.20, P< 0.001 ).4. LDTg/PPTg administration of huperzine A dose-dependently attenuates heroin cues-induced reinstatement of drug seekingMicroinjection of huperzine A into the LDTg/PPTg prior to presenting conditioned cues inhibited the reinstatement of heroin seeking. These data were analyzed with a one-way ANOVA, which revealed a significant main effect of treatment( F (2,21) = 12.67, P = 0.007 ). Subsequent Post hoc comparisons showed that the total active responses were significantly different between the huperzine A ( 1, 3μg/side ) and the vehicle treatments ( P =0.0003,P=0.0004).Conclusions:The present data demonstrated that findings as following:1. These data support that cholinergic transmission in?uences heroin self-administration and reinstatement.2. The present results indicate that cholinergic of projection neurons in the LDTg/PPTg play critical roles in the reinstatement of heroin seeking.3. The inhibitory effects of huperzine A, on heroin self-administration and reinstatement were found, providing the evidence for the huperzine A as an adjunctive therapy upon heroin addiction. |
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