Miaojun Lai

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.

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.

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.

The phosphodiesterase-4 inhibitor rolipram attenuates heroin-seeking behavior induced by cues or heroin priming in rats

Inhibition of phosphodiesterase-4 (PDE4), an enzyme that specifically hydrolyzes cyclic adenosine monophosphate (cAMP) increases intracellular cAMP/cAMP-response element binding protein (CREB) signaling. Activation of this signaling is considered as an important compensatory response that decreases motivational properties of drugs of abuse. However, it is not known whether PDE4 is involved in heroin seeking. Self-administration of heroin (50 μg/kg/infusion) was performed under the fixed ratio 1 (FR1) schedule for 14 d and then drug seeking was extinguished for 10 d. The progressive ratio schedule was used to evaluate the relative motivational value of heroin reinforcement. After training, the conditioned cue or heroin priming (250 μg/kg) was introduced for the reinstatement of heroin-seeking behavior. Pretreatment (i.p.) with rolipram (0.03-0.3 mg/kg), a prototypical, selective PDE4 inhibitor, failed to inhibit heroin self-administration under the FR1 schedule, but decreased the reward values under the progressive ratio schedule in a dose-dependent manner. In addition, rolipram decreased the reinstatement of heroin seeking induced by cues or heroin priming even at the lowest dose (0.03 mg/kg); in contrast, the highest dose (0.3 mg/kg) of rolipram was required to decrease sucrose reinforcement. Finally, the effects of rolipram on heroin-seeking behavior were correlated with the increases in expression of phosphorylated CREB in the nucleus accumbens. The study demonstrated that rolipram inhibited heroin reward and heroin-seeking behavior. The results suggest that PDE4 plays an essential role in mediating heroin seeking and that PDE4 inhibitors may be used as a potential pharmacotherapeutic approach for heroin addiction.

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.

Self-administration of propofol is mediated by dopamine D1 receptors in nucleus accumbens in rats.

As a widely used intravenous short-acting anesthetic, propofol is recently indicated by clinical and animal studies for its abuse potential, but the mechanism underlying propofol abuse is largely unknown. This study examined the contribution of dopamine receptor subtype (D1 and D2 receptors) and neuroanatomical locus (i.e. nuclear accumbens) in the maintenance of propofol self-administration in rats. After the acquisition and maintenance of self-administration of propofol (1.7 mg/kg/infusion) under a fixed ratio (FR1) schedule of reinforcement over 14 days, rats were treated by either intraperitoneal injection or intra-nucleus accumbens (NAc) injection of D1 receptor antagonist (SCH23390) or D2 receptor antagonists (spiperone and eticlopride) 10 min prior to the subsequent propofol self-administration. We demonstrated (i) systemic administration of SCH23390 (10, 30, 100 μg/kg, i.p.) dose-dependently decreased the rate of propofol-maintained self-administration, suggesting a critical role of the D1 receptor in mediating propofol self-administration; (ii) the blockade of the propofol self-administration by SCH23390 was specific since spiperone and eticlopride did not affect propofol self-administration and SCH23390 at these doses did not affect food-maintained responding under an FR5 schedule; (iii) intra-accumbenal injection of SCH23390 (2.5 μg/site) but not eticopride (3.0 μg/site) attenuated the propofol self-administration, localizing nuclear accumbal D1 receptors as a critical locus in the reinforcement of propofol. Together, these findings provide the first direct evidence that D1 receptors in nuclear accumbens play an important role in the maintenance of propofol self-administration.

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.

Microinjection of M(5) muscarinic receptor antisense oligonucleotide into VTA inhibits FosB expression in the NAc and the hippocampus of heroin sensitized rats.

ObjectiveTo investigate the effect of M5 muscarinic receptor subtype on the locomotor sensitization induced by heroin priming, and it’s effect on the FosB expression in the nucleus accumbens (NAc) and the hippocampus in the heroin sensitized rats.MethodsLocomotor activity was measured every 10 min for 1 h after subcutaneous injection of heroin. FosB expression was assayed by immunohistochemistry, and the antisense oligonucleotides (AS-ONs) targeting M5 muscarinic receptor was transferred with the lipofectin.ResultsMicroinjection of AS-ONs targeting M5 muscarinic receptor in the ventral tegmental area (VTA) blocked the expression of behavioral sensitization induced by heroin priming in rats. Meanwhile, the expression of FosB-positive neurons in either the NAc or the dentate gyrus (DG) of the hippocampus increased in heroin-induced locomotor sensitized rats. The enhancement of FosB-positive neurons in the NAc or DG could be inhibited by microinjection of M5 muscarinic receptor AS-ONs into the VTA before the heroin-induced locomotor sensitization was performed. In contrast, microinjection of M5 muscarinic receptor sense oligonucleotide (S-ONs) into the VTA did not block the expression of behavioral sensitization or the expression of FosB in the NAc or DG in the heroin sensitized rats.ConclusionBlocking M5 muscarinic receptor in the VTA inhibits the expression of heroin-induced locomotor sensitization, which is associated with the regulation of FosB expression in the NAc and hippocampus neurons. M5 muscarinic receptor may be a useful pharmacological target for the treatment of heroin addiction.摘要目的探讨 M5 毒蕈碱受体亚型对海洛因诱导的大鼠行为敏化以及敏化后大脑伏隔核 (NAc) 和海马中FosB蛋白表达的影响。方法建立海洛因诱导的大鼠行为敏化模型, 测定大鼠的自主活动量 (locomotor activity, LA), 观察 M5 毒蕈碱受体反义寡核苷酸 (M5AS-ONs) 对行为敏化表达的影响。 用免疫组化法测定大鼠NAc 及海马齿状回 (DG) FosB 蛋白表达。结果海洛因处理组与盐水处理组相比, 大鼠在 1 小时内的 LA 显著增 加, 表明这些大鼠已稳定建立了海洛因诱导的敏化。 中脑腹侧被盖区 (VTA) 中注射 M5 AS-ONs 能抑制大鼠 海洛因行为敏化的表达。 海洛因诱导的行为敏化大鼠中NAc及 DG 中的 FosB 免疫反应阳性神经元的表达增加, 而在 VTA 内注射 M5 AS-ONs 能明显抑制 NAc 及 DG 中 FosB 阳性神经元表达的增加; 但 VTA 中注射有义寡核 苷酸 (M5S-ONs) 不能明显抑制大鼠行为敏化的表达, 也不能抑制海洛因敏化大鼠NAc 和DG 中 FosB 蛋白的 表达。结论阻断 VTA 中 M5 毒蕈碱受体可抑制海洛因诱导的行为敏化的表达, 其机制可能和抑制大脑 NAc 和 DG 神经元中 FosB 蛋白的激活有关。 M5 毒蕈碱受体可作为改变海洛因行为学效应的有效药理学靶点之一。

Activation of AMPA receptor in the infralimbic cortex facilitates extinction and attenuates the heroin-seeking behavior in rats

Infralimbic cortex (IL) is proposed to suppress cocaine seeking after extinction, but whether the IL regulates the extinction and reinstatement of heroin-seeking behavior is unknown. To address this issue, the male SD rats were trained to self-administer heroin under a FR1 schedule for consecutive 14 days, then the rats underwent 7 daily 2h extinction session in the operant chamber. The activation of IL by microinjection PEPA, an allosteric AMPA receptor potentiator into IL before each of extinction session facilitated the extinction responding after heroin self-administration, but did not alter the locomotor activity in an open field testing environment. Other rats were first trained under a FR1 schedule for heroin self-administration for 14 days, followed by 14 days of extinction training, and reinstatement of heroin-seeking induced by cues was measured for 2h. Intra-IL microinjecting of PEPA at 15min prior to test inhibited the reinstatement of heroin-seeking induced by cues. Moreover, the expression of GluR1 in the IL and NAc remarkably increased after treatment with PEPA during the reinstatement. These finding suggested that activation of glutamatergic projection from IL to NAc shell may be involved in the extinction and reinstatement of heroin-seeking.