Huifen Liu

Role of acetylcholine transmission in nucleus accumbens and ventral tegmental area in heroin-seeking induced by conditioned cues

The involvement of cholinergic transmission in heroin self-administration and the reinstatement of heroin-seeking was examined in rats trained to nose-poke for i.v. heroin. Systemic treatment with physostigmine, an inhibitor of acetylcholinesterase, modestly reduced the acquisition and rate of heroin self-administration, and this suppression of heroin intake was reversed by pretreatment with scopolamine but not by mecamylamine. Following 10-14 days of self-administration, rats were left in the home environment for 14 days. Subsequently, rats were evaluated for extinction of nose-pokes during the first hour after being returned to the self-administration apparatus. One hour later a conditioned stimulus (house light, light in the nose-poke hole, sound of the infusion pump) was presented to initiate cue-induced reinstatement. Physostigmine produced a dose-dependent inhibition of cue-induced reinstatement, but only the dose of 0.5 mg/kg significantly decreased nose-poke responding in the extinction test. Chronic treatment with physostigmine (0.1 mg/kg) did not impair performance during acquisition of heroin self-administration. However, during a subsequent reinstatement test conducted in the absence of physostigmine pretreatment, heroin seeking was significantly below that of rats chronically pretreated with saline. To evaluate brain regions mediating the effects of systemic drug treatment on reinstatement, physostigmine was microinjected into the nucleus accumbens (NAc) or ventral tegmental area (VTA). Microinjection of physostigmine into the NAc prior to presenting conditioned cues inhibited the reinstatement of heroin-seeking, without affecting extinction responding. In contrast, microinjection of physostigmine into the VTA augmented the reinstatement induced by conditioned cues and extinction responding. Inactivation of either NAc or VTA by microinjecting tetrodotoxin blocked both extinction responding and cue-induced reinstatement. These data demonstrate that cholinergic transmission influences heroin self-administration and reinstatement. Moreover, cue-induced reinstatement was inhibited by physostigmine in the NAc and potentiated by cholinergic stimulation in the VTA.

Increased c-Fos expression in the medial part of the lateral habenula during cue-evoked heroin-seeking in rats

Conditioned environmental stimuli are known to be important determinants of drug seeking behavior. c-Fos, the protein product of the protooncogene c-Fos, is expressed in neurons when there are drug-associated cue-induced drug-seeking behaviour. Therefore, its expression could serve as a marker of regional neuronal activation. Using an extinction/reinstatement paradigm of relapse animal model, we trained Sprague-Dawley rats to nose-poke for i.v. heroin (0.05 mg/kg/infusion) either daily for 4h or 25 infusions for 14 consecutive days. We then tested these animals for cue-evoked heroin-seeking behavior after abstinence from self-administration of heroin for 14 days. Expression of c-Fos was examined in the lateral habenula (LHb), a region important for conveying information between the limbic forebrain and midbrain. Findings showed that heroin-associated conditioned stimuli could induce robust heroin-seeking behavior that was associated with increased c-Fos immunoreactivity in the medial part of the LHb. This observation suggests the involvement of the LHb in mediating drug cue-induced heroin-seeking behavior after abstinence from self-administration of heroin.

Electroacupuncture attenuates morphine withdrawal signs and c-Fos expression in the central nucleus of the amygdala in freely moving rats

Experimental efforts for understanding the mechanisms of electroacupuncture (EA) for opiate addiction are partially hampered by restraint stress. In unrestrained animals, it is difficult to perform EA stimulation at acupuncture points frequently selected on the four limbs. The present study was performed to evaluate the effect of EA at the acupuncture point Shen-Shu (BL.23) on morphine withdrawal signs and c-Fos expression of the amygdala in freely moving rats or restrained rats. We applied immunohistochemistry to detect c-Fos-positive nuclei. Corticosterone levels and behavioral responses were measured during EA stimulation. The needles were bilaterally inserted and fixed at BL.23, and 100-Hz electric stimulation was conducted 30 min before naloxone-precipitated withdrawal. In both freely moving rats and restrained rats, EA significantly reduced the signs of morphine withdrawal. Notably, EA stimulation in freely moving rats attenuated c-Fos expression in the central nucleus of the amygdala while EA in restrained animals increased this response. In addition, the restrained rats emitted greater levels of vocalization and facial expression than freely moving rats during EA stimulation. Corticosterone levels were also significantly higher in restrained animals after EA stimulation. The new EA paradigm demonstrated in the present study might help the analysis of certain physiological responses induced by EA that would otherwise have been hindered by restraint stress.

DBS of nucleus accumbens on heroin seeking behaviors in self-administering rats

BACKGROUND Surgical ablation of select brain areas has been frequently used to alleviate psychological dependence on opiate drugs in certain countries. However, ablative brain surgery was stopped in China in 2004 due to the related ethical controversy and possible side effects. Deep brain stimulation (DBS), a less invasive, reversible and adjustable process of neuromodulation, was adopted to attenuate relapses in studies of drug addiction. METHODS Preclinical experiments were designed to assess the long-term effects of DBS of the nucleus accumbens (NAc) on cue- and heroin-induced reinstatement of drug seeking behaviors. After a rat self-administration model of heroin relapse was established, DBS was administered bilaterally or unilaterally to the NAc core through concentric bipolar electrodes. A 1-h long continuous stimulation (130 Hz, 100 μs, 0-150 μA) was given daily for 7 days during the abstinence session. Drug seeking behaviors were elicited by conditioned cues or a small dose of heroin. RESULTS 75 μA and 150 μA bilateral NAc DBS attenuated cue- and heroin-induced reinstatement of drug seeking, and unilateral DBS of the right NAc achieved effects almost equivalent to bilateral DBS. Additional experiments showed that DBS had no long-term influence on locomotor activity and spatial learning and retention capabilities in Morris water maze tasks. Subsequent immunohistochemistry measurements revealed that the behavioral consequences were associated with a significant increase in the expression of pCREB and a reduction in the expression of ΔFosB in the NAc. CONCLUSIONS These findings indicate that the NAc DBS could be an effective and safe therapeutic option for preventing relapse to heroin addiction.

Motivation of heroin-seeking elicited by drug-associated cues is related to total amount of heroin exposure during self-administration in rats

Conditioned stimuli (CSs) previously associated with heroin are critically involved in activating long-lasting relapse and compulsive drug seeking. This study examined the magnitude of heroin seeking induced by drug-related cues in relation to the total amount of drug exposure during training. Five groups of male Sprague-Dawley rats (n=6/group) were trained by the nose-poking response to self-administer different doses of heroin (0, 0.01, 0.025, 0.05, and 0.1 mg/kg per infusion respectively, one 4-h session daily, limited to 25 infusions per session) under an identical progressive ratio schedule with gradual incremental response requirements. All the rats established stable heroin self-administration within 14 days of self-administration training, and the time needed to obtain all the 25 heroin infusions decreased across sessions. After 14 days of abstinence, heroin seeking elicited by contextual cues (self-administration chamber) or discrete contingent CSs previously associated with heroin infusions was measured in two consecutive 1-h test phases. During both test phases, the rats trained with heroin even at the lowest dose (0.01 mg/kg) showed higher active responses than saline controls, and the active responses were also higher in rats trained with doses of 0.025, 0.05, and 0.10 mg/kg in comparison with those trained with a dose of 0.01 mg/kg. There was no observable dose-dependence increase of responses at doses above 0.025 mg/kg. The results suggested that an increased motivation to seek heroin induced by drug-related cues is associated with the total amount of heroin intake.

A Heroin Addiction Severity-Associated Intronic Single Nucleotide Polymorphism Modulates Alternative Pre-mRNA Splicing of the μ Opioid Receptor Gene OPRM1 via hnRNPH Interactions

Single nucleotide polymorphisms (SNPs) in the OPRM1 gene have been associated with vulnerability to opioid dependence. The current study identifies an association of an intronic SNP (rs9479757) with the severity of heroin addiction among Han-Chinese male heroin addicts. Individual SNP analysis and haplotype-based analysis with additional SNPs in the OPRM1 locus showed that mild heroin addiction was associated with the AG genotype, whereas severe heroin addiction was associated with the GG genotype. In vitro studies such as electrophoretic mobility shift assay, minigene, siRNA, and antisense morpholino oligonucleotide studies have identified heterogeneous nuclear ribonucleoprotein H (hnRNPH) as the major binding partner for the G-containing SNP site. The G-to-A transition weakens hnRNPH binding and facilitates exon 2 skipping, leading to altered expressions of OPRM1 splice-variant mRNAs and hMOR-1 proteins. Similar changes in splicing and hMOR-1 proteins were observed in human postmortem prefrontal cortex with the AG genotype of this SNP when compared with the GG genotype. Interestingly, the altered splicing led to an increase in hMOR-1 protein levels despite decreased hMOR-1 mRNA levels, which is likely contributed by a concurrent increase in single transmembrane domain variants that have a chaperone-like function on MOR-1 protein stability. Our studies delineate the role of this SNP as a modifier of OPRM1 alternative splicing via hnRNPH interactions, and suggest a functional link between an SNP-containing splicing modifier and the severity of heroin addiction.

Positive association between--1021TT genotype of dopamine beta hydroxylase gene and progressive behavior of injection heroin users.

By balancing the ratios of dopamine and norepinephrine, dopamine beta hydroxylase (DBH) plays an important role in brain reward circuit that is involved with behavioral effects of heroin addiction. DBH -1021C/T (rs1611115) is a functional variant with strong correlation with plasma DBH activity and several nerval and psychic disorders. In the present study, we have collected 333 male cases with heroin addiction and 200 male healthy controls to explore the role of -1021C/T in heroin addiction. There is no evidence of association between -1021C/T and heroin addiction on both genotype and allele levels (P>0.05). In the injection subgroup of cases, -1021TT carriers have longer heroin addiction time (P<0.001) and higher dosage of self-administered heroin (P=0.045) than carriers with -1021CC or -1021CT, suggesting that patients with TT genotype are likely to have more progressive style of heroin users with injection route. In conclusion, our results support -1021TT genotype may be implicated with a more progressive nature of heroin addiction, although DBH -1021C/T is unlikely to be involved in the risk of heroin addiction.

Low dose of heroin inhibits drug-seeking elicited by cues after prolonged withdrawal from heroin self-administration in rats

Environmental stimuli and conditioned cues associated with heroin can induce drug-seeking behavior, but how heroin lapse interacts with cues is unclear. Rats were trained to nose-poke for i.v. heroin for 14 days and then tested for heroin seeking after withdrawal from heroin self-administration. Heroin seeking induced by cues persisted over several weeks after withdrawal, and the responding was not easily extinguished after 4 weeks withdrawal. A single injection of heroin (250 μg/kg) enhanced the responding at early stage of withdrawal, but a low dose of heroin (50, 250 μg/kg) suppressed the responding induced by contextual or conditioned cues at 4 weeks of withdrawal. The results suggest that prolonged withdrawal may increase the risk of relapse to heroin seeking.

Vagus nerve stimulation inhibits heroin-seeking behavior induced by heroin priming or heroin-associated cues in rats

Vagus nerve stimulation has been used for the treatment of neuropsychiatric disorders, such as epilepsy. However, little is known whether it is also effective for the treatment of heroin dependence, in particular for relapse to heroin seeking. In the present study, we investigated the effects of vagus nerve stimulation on reinstatement (relapse) of heroin-seeking behavior induced by heroin priming or heroin-associated cues. The rats were trained for heroin self-administration for 14days and followed by extinction training in which heroin was replaced by saline and heroin-associated cues were turned off. In addition, animals were also received daily electric stimulation of vagus nerve (30Hz, pulse width of 0.5ms, 0.5mA (low-intensity) or 1mA (high-intensity); 30s on, 5min off; 10 continuous cycle per day) or false stimulation during extinction training. We found that such vagus nerve stimulation significantly inhibited heroin priming (0.25mg/kg, s.c.) - or heroin-associated conditioned cue-induced reinstatement of drug-seeking behavior, when compared to false stimulation control. Further, such a behavioral inhibition was correlated to a reduction in the expression of FosB and an increase in the expression of phosphorylation of cAMP response element binding protein (p-CREB) in nucleus accumbens. The data suggest that vagus nerve stimulation may inhibit heroin- or heroin cue-induced relapse, possibly by regulation of the expression of Fos and CREB in nucleus accumbens.

Galantamine attenuates the heroin seeking behaviors induced by cues after prolonged withdrawal in rats

BACKGROUND AND OBJECTIVE Evidence shows that acetylcholinergic transmission in the ventral tegmental area (VTA) or nucleus accumbens (NAc) plays an important role in heroin-seeking induced by cues. Cholinergic modulation of VTA neurons arises from the lateral dorsal tegmental nucleus (LDT). The present studies investigated the effect of systemic or intra- LDT administration of galantamine, an inhibitor of acetylcholinesterase, on heroin-seeking induced by cues. METHODS Rats were trained to self-administer heroin for 12 days, underwent extinction training for 12 days followed by two weeks in their home cages. Then the conditioned cues were introduced for the reinstatement of heroin-seeking. RESULTS The reinstatement of heroin-seeking induced by cues was attenuated by the administration of galantamine (0, 0.3, 1 or 3mg/kg, i.p.) in a dose-dependent manner. In contrast, galantamine only at the dose of 3mg/kg could inhibit the reinstatement of sucrose-seeking. Galantamine at those doses failed to alter the locomotor activity in heroin-withdrawn rats. The inhibition of drug-seeking by galantamine was reversed by pretreatment with scopolamine (0.5mg/kg) but not with mecamylamine (3mg/kg) or scopolamine methobromide (1mg/kg). Moreover, the microinjection of galantamine into the LDT blocked cue-induced heroin-seeking, while the microinjection of scopolamine into the LDT reversed the inhibitory effect of galantamine on drug-seeking behavior. CONCLUSION The results suggest that cholinergic transmission in the LDT may play a critical role in heroin-seeking behavior induced by cues and that galantamine may have the beneficial effect of blocking heroin-seeking behavior, which is mediated through its actions on the muscarinic receptors.