Roles of forced nicotine exposure and Comt gene disruption in the development of addiction-related behavioural and neurochemical changes in mice

Activation of midbrain dopamine systems is thought to be critically involved in the addictive properties of abused substances. Drugs of abuse increase dopamine release in the nucleus accumbens and dorsal striatum, which are the target areas of mesolimbic and nigrostriatal dopamine pathways, respectively. Dopamine release in the nucleus accumbens is thought to mediate the attribution of incentive salience to rewards, and dorsal striatal dopamine release is involved in habit formation. In addition, changes in the function of prefrontal cortex (PFC), the target area of mesocortical dopamine pathway, may skew information processing and memory formation such that the addict pays an abnormal amount of attention to drug-related cues. In this study, we wanted to explore how long-term forced oral nicotine exposure or the lack of catechol-O-methyltransferase (COMT), one of the dopamine metabolizing enzymes, would affect the functioning of these pathways. We also wanted to find out how the forced nicotine exposure or the lack of COMT would affect the consumption of nicotine, alcohol, or cocaine. First, we studied the effect of forced chronic nicotine exposure on the sensitivity of dopamine D2-like autoreceptors in microdialysis and locomotor activity experiments. We found that the sensitivity of these receptors was unchanged after forced oral nicotine exposure, although an increase in the sensitivity was observed in mice treated with intermittent nicotine injections twice daily for 10 days. Thus, the effect of nicotine treatment on dopamine autoreceptor sensitivity depends on the route, frequency, and time course of drug administration. Second, we investigated whether the forced oral nicotine exposure would affect the reinforcing properties of nicotine injections. The chronic nicotine exposure did not significantly affect the development of conditioned place preference to nicotine. In the intravenous self-administration paradigm, however, the nicotine-exposed animals self-administered nicotine at a lower unit dose than the control animals, indicating that their sensitivity to the reinforcing effects of nicotine was enhanced. Next, we wanted to study whether the Comt gene knock-out animals would be a suitable model to study alcohol and cocaine consumption or addiction. Although previous work had shown male Comt knock-out mice to be less sensitive to the locomotor-activating effects of cocaine, the present study found that the lack of COMT did not affect the consumption of cocaine solutions or the development of cocaine-induced place preference. However, the present work did find that male Comt knock-out mice, but not female knock-out mice, consumed ethanol more avidly than their wild-type littermates. This finding suggests that COMT may be one of the factors, albeit not a primary one, contributing to the risk of alcoholism. Last, we explored the effect of COMT deficiency on dorsal striatal, accumbal, and prefrontal cortical dopamine metabolism under no-net-flux conditions and under levodopa load in freely-moving mice. The lack of COMT did not affect the extracellular dopamine concentrations under baseline conditions in any of the brain areas studied. In the prefrontal cortex, the dopamine levels remained high for a prolonged time after levodopa treatment in male, but not female, Comt knock-out mice. COMT deficiency induced accumulation of 3,4-dihydroxyphenylacetic acid, which increased further under levodopa load. Homovanillic acid was not detectable in Comt knock-out animals either under baseline conditions or after levodopa treatment. Taken together, the present results show that although forced chronic oral nicotine exposure affects the reinforcing properties of self-administered nicotine, it is not an addiction model itself. COMT seems to play a minor role in dopamine metabolism and in the development of addiction under baseline conditions, indicating that dopamine function in the brain is well-protected from perturbation. However, the role of COMT becomes more important when the dopaminergic system is challenged, such as by pharmacological manipulation.

A susceptibilidade à nicotina e etanol é afetada pela exposição à nicotina, fumaça de cigarro e etanol durante o período gestacional: alterações comportamentais e eletrofisiológicas no período pós-natal de camundongos

Uma questão particularmente relevante é o fato de exposições precoces a drogas de abuso durante o desenvolvimento potencialmente aumentarem a susceptibilidade a estas drogas posteriormente durante o desenvolvimento. No presente estudo utilizando estudos comportamentais e eletrofisiológicos, investigamos efeitos tardios da exposição de camundongos à fumaça de cigarro, à nicotina e ao etanol durante o período que corresponde à gestação em humanos. Para tal, esta tese foi dividida em 2 estudos. No Estudo 1, submetemos camundongos durante o período que corresponde à gestação de humanos à fumaça de cigarro e/ou etanol visando investigar se a estas drogas de abuso, separadamente ou quando combinadas, programam maior susceptibilidade aos efeitos da nicotina durante a adolescência (PN30) ou idade adulta (PN90). Para avaliar a susceptibilidade, utilizamos 3 testes: campo aberto (CA), preferencia pela nicotina (PPN) e preferencia condicionada por lugar (CPP). No Estudo 2, os animais foram expostos a nicotina durante o período gestacional e, no período que corresponde à infância (PN9 a PN20), fatias de cérebro contendo o núcleo tegumental laterodorsal (LDT) foram expostas a etanol. Este núcleo foi escolhido uma vez que estudos recentes indicam sua participação em mecanismos de toxicodependência. Foram realizados registros eletrofisiológicos de uma única célula. No Estudo 1, identificamos maior sensibilidade para os efeitos da reexposição à nicotina na adolescência quando comparada com a idade adulta . Em animais testados no CA durante a adolescência, a nicotina foi capaz de causar aumento da atividade locomotora nos animais controle, previamente expostos à fumaça de cigarro e ao etanol. Contudo, em animais expostos à fumaça combinada com etanol, não houve aumento da locomoção. Na idade adulta, a nicotina causou um aumento da atividade locomotora no CA somente nos animais expostos à fumaça de cigarro. Quanto ao CPP, a exposição prévia à fumaça de cigarro e ao etanol causaram aumento da resposta condicionada à nicotina em fêmeas adolescentes. Nos animais previamente expostos à combinação entre fumaça de cigarro e etanol, a resposta condicionada à nicotina não atingiu significância estatística. Não houve alterações na idade adulta. A exposição a fumaça de cigarro e/ou etanol não afetou a PPN. No Estudo 2, os dados eletrofisiológicos mostraram que a exposição pré-natal à nicotina foi capaz de alterar as correntes de despolarização basais e o potencial de repouso de células do LDT A nicotina também foi capaz de alterar as respostas deste núcleo ao etanol reduzindo as correntes de despolarização e aumentando, embora que não de forma significativa, as correntes inibitórias. De acordo com estes dados, injurias causadas pela exposição à fumaça do cigarro, à nicotina isoladamente, e ao etanol durante o desenvolvimento são capazes de perdurar por um logo tempo na vida do individuo, alterando as respostas a comportamentais e celulares a uma exposição tardia à nicotina e ao etanol.

特异突触可塑性干扰肽在小鼠吗啡条件化位置偏爱表达过程中的作用

在作为成瘾检测手段的条件化位置偏爱模型中,环境背景和成瘾药物间的关联性学习起着关键的作用.突触可塑性作为学习记忆可能的物质基础,在药物成瘾方面的研究也越来越多,但其表现形式,长时程增强(LTP)或者长时程抑制(LTD)在成瘾过程中所发挥的具体作用尚不得而知.因此,本文利用生物信息学手段,设计并合成了旨在分别阻断LTP和LTD的干扰肽,研究其对小鼠吗啡条件化位置偏爱的影响.结果发现,干扰肽Pep-A2和Pep-A3能够分别特异地阻断海马CA1区的LTP和LTD,在测试前尾静脉注射具有穿膜特性的LTP/LTD特异性干扰肽(Tat-A2/Tat-A3),均能阻断或损伤吗啡诱导的条件化位置偏爱的表达.此发现提示我们,LTP和LTD在成瘾性异常记忆的过程中均发挥着重要的作用.

慢性吗啡给予、戒断及再给药过程中大鼠海马感觉门控的动态变化

药物成瘾被认为是药物长期作用于脑而产生的一种慢性复吸性脑疾病,长期反复的药物(如吗啡)滥用会导致一系列严重后果,如药物依赖、药物耐受、强迫性药物寻求等.本实验利用条件化位置偏好(conditioned place preference,CPP)模型来检测大鼠对吗啡依赖和心理渴求等过程;采用双声刺激听觉诱发电位来研究大鼠在慢性吗啡给予、戒断以及再给药过程中海马感觉门控(N40)的动态变化.吗啡组大鼠注射吗啡(10mg/kg,i.p.)12d,经历第一次戒断12d,再次注射吗啡(2.5mg/kg,i.p.)1d,之后经历第二次戒断2d;对照组大鼠注射同体积生理盐水,其余实验条件与吗啡组相同.CPP实验表明,这种药物给予方法促使大鼠对吗啡产生药物依赖和心理渴求.双声刺激诱发电位实验表明,吗啡组大鼠在吗啡给予期间海马感觉门控受到损伤;第一次戒断期的第1～2天海马感觉门控能力减弱,第3天增强,第4～12天逐渐恢复到正常水平;再次给予吗啡后海马感觉门控能力与对照组相比显著降低,并且随后2d的戒断期内海马感觉门控能力也一直保持较低水平,表明再次给药使大鼠海马感觉门控对吗啡更加敏感化.结果提示,长期反复的吗啡给予及再给药干扰了海马的感觉门控能力,吗啡成瘾对大脑可能产生长期影响.

特异性干扰肽对海马突触可塑性的影响及其功能研究

在哺乳动物复杂的神经网络中，突触是信息传递的枢纽，其突触传递效能的持续性变化被称为突触可塑性（synaptic plasticity）。长时程增强（long-term potentiation，LTP）和长时程抑制（long-term depression，LTD）现象是两种经典的突触可塑性形式，被视作学习和记忆可能的物质基础，得到了广泛地关注。其中，海马CA1区谷氨酸能突触处的LTP和LTD目前研究得最为广泛。 α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid（AMPA）受体作为介导兴奋性谷氨酸能突触基础传递的主要受体，是海马CA1区LTP和LTD正常表达的必要条件。近期的研究表明，AMPA受体通过胞吞、胞吐及侧向移动等方式在细胞膜和细胞内进行着持续地循环。因此，通过调节AMPA受体的上、下膜，进而影响突触后膜上AMPA受体的数量，便能对LTP和LTD产生影响。在本研究中，我们利用生物信息学的手段，以AMPA受体为靶点，设计出了旨在特异阻断LTP或LTD的多肽。运用离体脑片全细胞记录方式，在海马CA1区证明了干扰肽Pep-A2能够特异地阻断LTP而不影响LTD，Pep-A3能够特异地阻断LTD而不影响LTP。并初步探究了其关键的作用位点，为进一步理解LTP和LTD具体的分子机理打下了基础。成瘾作为异常的学习记忆过程，势必涉及到突触可塑性的变化。而特异性地阻断LTP和LTD，对药物成瘾效果的影响却鲜有报道（Wang YT，2007）。在另一部分工作中，我们采用穿膜肽Tat-A2和Tat-A3，在吗啡条件化位置偏爱（morphine conditioned place preference，morphine CPP）模型小鼠的测试前进行系统给药，结果发现两种干扰肽均能阻断或损伤其CPP的表达过程。这一现象，提示我们LTP和LTD在条件化位置偏爱的表达过程中都是不可或缺的，同时也为人们更好地理解成瘾过程的机理，及开发专一有效的治疗药物提供了新的思路。

吗啡成瘾相关学习记忆的脑机制

重复使用吗啡将导致吗啡成瘾，其主要表现如依赖、耐受、敏感化以及吗啡停用后的戒断反应。其核心特征是强迫性吗啡使用：即成瘾者失去了对药物寻觅和摄取的控制。药物成瘾是一个复杂的生物学过程，近年来的研究表明学习记忆参与药物成瘾过程。学习记忆和药物成瘾都受到相似的神经营养因子，递质释放与转运的调控，它们都受到cAMP，CREB等调控因子的调控。研究发现在与成瘾相关的线索，如用药有关的人物、地点或暗示等，都可能恢复觅药和用药行为。当把成瘾相关的线索呈现给戒断中的人时，可以发现这些人表现异常，如心率、呼吸加快，血压升高，并表现明显的渴求行为，前额叶在这种线索导致的渴求行为中起重要作用。以前的研究证实条件化位置偏好(conditioned place preference，CPP)是一个很好的模型来研究环境线索在成瘾中作用，可以用来分析检测成瘾行为。因此，在实验一、二中，采用CPP模型来研究吗啡相关的学习记忆与胆碱系统，以及蛋白质合成的关系；实验三研究了前额叶和海马在吗啡成瘾和戒断过程中谷氨酸(GLU)和γ-氨基丁酸(γ-GABA)含量的变化。 (1)：学习和记忆依赖于多种递质的共同作用，不同的递质在不同的学习记忆中的作用也不一样。大量的研究表明胆碱系统在学习记忆中起重要作用，胆碱系统抑制剂可以导致学习记忆障碍。在本实验中，我们研究东莨菪碱(经典的抗胆碱能药物，影响记忆的获取过程)对吗啡以及食物相关的线索的记忆的影响，研究抑制胆碱系统对这两种线索相关的奖赏性学习记忆的影响。采用腹腔注射东莨菪碱，三个不同的剂量(0.5、1、2mg/kg)，在给吗啡(40 mg/kg)或者食物之前30分钟给东莨菪碱，连续给药4天。结果显示，除了0.5 mg/kg的东莨菪碱不能抑制食物导致的CPP外，其他剂量的东莨菪碱都明显抑制食物导致CPP。但是东莨菪碱并不能抑制吗啡导致的CPP，而且，2.0 mg/kg东莨菪碱强化了吗啡线索相关的学习记忆。这种结果表明吗啡导致的奖赏性学习和普通的普通记忆存在不同的机制。 (2)：记忆的形成需要蛋白质的合成，特别是长时程记忆。在记忆形成的不同阶段，蛋白质的合成对记忆的影响不一样，在学习前后较短的时间内给蛋白质合成抑制剂可以有效的抑制长时程记忆的形成。学习记忆参与吗啡成瘾过程，那么抑制蛋白质的合成是否也能抑制吗啡成瘾相关的学习记忆呢？本实验中，采用环己酰亚胺(蛋白质合成抑制剂，抑制记忆的巩固)来研究抑制蛋白质合成对吗啡线索相关的学习记忆的影响。腹腔给环己酰亚胺，剂量30mg/kg，给药时间为给吗啡前30分钟，同时，后30分钟以及后2小时。实验发现在给吗啡前后30分钟内给环己酰亚胺可以削弱对吗啡相关的线索的记忆，但是不能完全抑制这种记忆的形成，而且在吗啡后两小时给环己酰亚胺基本上对这种记忆没有抑制作用。这种结果表明吗啡相关的联合型学习记忆的形成并不完全依赖于蛋白质的合成，脑内还存在其他的途径来参与这种记忆过程。 (3)：GLU和GABA参与许多学习记忆过程，同样它们在吗啡成瘾过程中也起重要的作用。前额叶和海马在记忆中都起到重要的作用，而且它们在成瘾记忆中也扮演重要角色。那么在成瘾及戒断过程中，前额叶和海马中GLU和GABA会有什么变化呢？本实验利用高效液相色谱－紫外分析法分析在吗啡成瘾及戒断过程中前额叶和海马中总GLU和GABA的动态变化。实验发现在吗啡成瘾及戒断过程中，GABA和GLU在前额叶和海马中的总含量都没有显著的变化，尽管在给药和戒断中，这两种递质都下调然后逐渐上升，但是跟对照组比较都不显著。但是比较吗啡组之间的变化，可以看出前额叶GLU在给药及戒断过程中有比较显著的变化。该结果提示在吗啡成瘾和戒断过程中，GLU和GABA的总量并没有发生显著的改变。结合前人的研究我们认为，这两种递质在成瘾和戒断过程中，它们的合成，释放以及在突触间的转运等各个环节都受到影响，从而参与吗啡成瘾。

鸡胚吗啡用药对学习记忆和成瘾易感性的影响及其受体机制

Prenatal morphine exposure affects neural development of fetus by impairing learning and memory, and increasing susceptibility to morphine abuse. Because nervous systems have different developmental characteristics during different developmental stages, administration of morphine at different stages also has different effects on learning, memory, and susceptibility to morphine. Due to the precise developmental processes of neurotransmitter systems in chick embryo’s brain, and unique superiority of chick embryo model, the purpose of the present studies was to explore critical periods correlated to the memory impairment and the increasing susceptibility to morphine, via one-trial passive avoidance and conditioned place preference as behavior models. Then the possible roles of mu and delta opioid receptors as the possible mechanism were analyzed. Experiment 1 showed that injecting low dose of morphine (1 mg/kg) during the period embryonic 5 to 8 significantly impaired the function of learning and memory, worse than any other periods of the same treatment. Experiment 2 showed that injecting low dose of morphine during the period embryonic 17 to 20 significantly increased the susceptibility to morphine in the new-born chicks. The affected chicks acquired the morphine conditioned place preference more quickly, and maintained it much longer. Experiment 3 showed that during E5-8, injecting delta receptor antagonist naltrindole reversed the learning and memory impairment caused by morphine while delta receptor agonist DPDPE impaired learning and partial memory function. On the other hand, mu opioid receptors had little effect. As for E17-20, given naloxonazine can reverse the increases of susceptibility to morphine, and the mu receptor agonist DAGO cause the increases of susceptibility to morphine. Delta receptors have no effect. The above results demonstrated that prenatal morphine expousure at different developmental periods of chick embryo caused different influences on memory and susceptibility to morphine. That is, E5-8 is the critical period correlate to memory impairment; and E17-20 is the critical period correlate to susceptibility to morphine. Delta receptors were critical in learning and memory impairment while mu receptors in susceptibility.

吗啡相关情境线索的形成及效应表达的神经基础——海马与基底外侧杏仁核在其中的作用

Drug-associated cue-induced relapse to drug seeking causes most difficulties of therapy for drug addiction. Addicts are exposed to two forms of environmental stimuli during drug-taking: contextual stimuli (e.g. a house in which the drug is consumed) and discrete stimuli (DS, e.g. a crack pipe or a syringe for drug). These stimuli become contextual cues and discrete cues, respectively. The incentive value of contextual cues plays a great role in opiates relapse. Compared with drug self-administration model, conditioned place preference (CPP) reflects the approach behavior for drug cues, not concerned with acquisition of operant behaviors. The present study aimed to investigate the role of basolateral amygdala (BLA) and hippocampus in the effect of opiates-related contextual cues using CPP model. Establishing DS-dependent or contextual cues-dependent CPP, the effect of BLA or hippocampus inactivation prior to training phase on acquisition of contextual cues-opiates association was evaluated. Inactivation prior to test phase was used to evaluate roles of BLA and hippocampus in expression of contextual cues-dependent morphine CPP. The main results were as follows: Inactivation of BLA or dorsal hippocampus selectively impaired acquisition of contextual cue-dependent CPP, but inactivation of ventral hippocampus had no impact on acquisition of either DS-dependent or contextual cue-dependent morphine CPP. Inactivation of BLA selectively inhibited expression of contextual cue-depended CPP. Inactivation of ventral hippocampus inhibited expression of both DS-dependent and contextual cue-dependent morphine CPP. These results suggest that BLA and dorsal hippocampus contribute to contextual cue association with opiates but not DS-opiates association. BLA and ventral hippocampus play important roles in incentive value of contextual cues. The present study provides more information for the neurological substrates underlying contextual cues associated with opiates.

影响吗啡动物行为效应的因素及其机制

Credible and stable animal behavioral models are necessary to research the mechanisms of addiction in vivo, especially to study the relationship between memory or stress and drug addiction, which has been one of the focuses in this field. So the object of this study was to observe the influences of several factors on the behavioral effects of morphine shown in the paradigms of conditioned place preference (CPP) and locomotor activity (LA), and to explore the effects of adrenalectomy on LA induced by morphine in rats. In addition, the cortexes of rats were examined, which were exposed to chronic administration of several doses of morphine with or without foot shock. Moreover, a new behavioral model was built to quantify the motivation of drug seeking. The results showed that CPP was more sensitive to low dose of morphine than to high dose. The period of experiment could be shortened by increasing the training times everyday, whereas in this way the dose of morphine should be low enough to avoid the impact between the near two exposures to morphine. Effects of chronic administration of morphine on LA in rats were dose- and time- dependent, which supplied evidence to choose parameters in other behavioral models. The results obtained by the simplified LA paradigm showed that hyperactivity of low dose of morphine following hypoactivity, and naloxone had no effects on LA but blocked the locomotion effects of morphine. Obvious effects of morphine on LA of rats might depend on a reasonable level of plasma corticosterone, which may determine individual vulnerability to drug addiction. Stress may also potentiate the vulnerability by aggravating damage to cortex of rats induced by drug dose-dependently, which is suggested by the results of histological examination. The result that frontal and temporal cortexes and hippocampus were injured suggests that there may be a close relationship between memory and drug addiction. It was showed that the new behavioral model on the basis of Morris water maze might be used to quantify the motivation of drug-craving.

Enhanced rewarding properties of morphine, but not cocaine, in beta(arrestin)-2 knock-out mice.

The reinforcing and psychomotor effects of morphine involve opiate stimulation of the dopaminergic system via activation of mu-opioid receptors (muOR). Both mu-opioid and dopamine receptors are members of the G-protein-coupled receptor (GPCR) family of proteins. GPCRs are known to undergo desensitization involving phosphorylation of the receptor and the subsequent binding of beta(arrestins), which prevents further receptor-G-protein coupling. Mice lacking beta(arrestin)-2 (beta(arr2)) display enhanced sensitivity to morphine in tests of pain perception attributable to impaired desensitization of muOR. However, whether abrogating muOR desensitization affects the reinforcing and psychomotor properties of morphine has remained unexplored. In the present study, we examined this question by assessing the effects of morphine and cocaine on locomotor activity, behavioral sensitization, conditioned place preference, and striatal dopamine release in beta(arr2) knock-out (beta(arr2)-KO) mice and their wild-type (WT) controls. Cocaine treatment resulted in very similar neurochemical and behavioral responses between the genotypes. However, in the beta(arr2)-KO mice, morphine induced more pronounced increases in striatal extracellular dopamine than in WT mice. Moreover, the rewarding properties of morphine in the conditioned place preference test were greater in the beta(arr2)-KO mice when compared with the WT mice. Thus, beta(arr2) appears to play a more important role in the dopaminergic effects mediated by morphine than those induced by cocaine.

Defining and assessing animal pain

The detection and assessment of pain in animals is crucial to improving their welfare in a variety of contexts in which humans are ethically or legally bound to do so. Thus clear standards to judge whether pain is likely to occur in any animal species is vital to inform whether to alleviate pain or to drive the refinement of procedures to reduce invasiveness, thereby minimizing pain. We define two key concepts that can be used to evaluate the potential for pain in both invertebrate and vertebrate taxa. First, responses to noxious, potentially painful events should affect neurobiology, physiology and behaviour in a different manner to innocuous stimuli and subsequent behaviour should be modified including avoidance learning and protective responses. Second, animals should show a change in motivational state after experiencing a painful event such that future behavioural decision making is altered and can be measured as a change in conditioned place preference, self-administration of analgesia, paying a cost to access analgesia or avoidance of painful stimuli and reduced performance in concurrent events. The extent to which vertebrate and selected invertebrate groups fulfil these criteria is discussed in light of the empirical evidence and where there are gaps in our knowledge we propose future studies are vital to improve our assessment of pain. This review highlights arguments regarding animal pain and defines criteria that demonstrate, beyond a reasonable doubt, whether animals of a given species experience pain.

A behavioral study of alpha-1b adrenergic receptor knockout mice: increased reaction to novelty and selectively reduced learning capacities.

Knockout mice lacking the alpha-1b adrenergic receptor were tested in behavioral experiments. Reaction to novelty was first assessed in a simple test in which the time taken by the knockout mice and their littermate controls to enter a second compartment was compared. Then the mice were tested in an open field to which unknown objects were subsequently added. Special novelty was introduced by moving one of the familiar objects to another location in the open field. Spatial behavior and memory were further studied in a homing board test, and in the water maze. The alpha-1b knockout mice showed an enhanced reactivity to new situations. They were faster to enter the new environment, covered longer paths in the open field, and spent more time exploring the new objects. They reacted like controls to modification inducing spatial novelty. In the homing board test, both the knockout mice and the control mice seemed to use a combination of distant visual and proximal olfactory cues, showing place preference only if the two types of cues were redundant. In the water maze the alpha-1b knockout mice were unable to learn the task, which was confirmed in a probe trial without platform. They were perfectly able, however, to escape in a visible platform procedure. These results confirm previous findings showing that the noradrenergic pathway is important for the modulation of behaviors such as reaction to novelty and exploration, and suggest that this is mediated, at least partly, through the alpha-1b adrenergic receptors. The lack of alpha-1b adrenergic receptors in spatial orientation does not seem important in cue-rich tasks but may interfere with orientation in situations providing distant cues only.

Effets motivationnels des cannabinoïdes dans un modèle animal de la schizophrénie

Depuis quelques décennies, la consommation de cannabis et son usage thérapeutique sont le sujet de nombreux débats. Le cannabis est la drogue illicite la plus consommée au monde et cette consommation se trouve dix fois plus élevée chez les patients atteints de schizophrénie que dans la population générale. L’hypothèse d’une automédication initialement proposée afin d’expliquer la consommation élevée de cannabis chez les patients atteints de schizophrénie est maintenant remise en question. En effet, les rapports indiquant une aggravation des symptômes plutôt qu’une amélioration suite à une consommation à long terme sont de plus en plus nombreux. Sachant que le cannabis peut induire des effets soit plaisants soit aversifs, la question se pose à savoir si une prédominance de la valence motivationnelle positive ou une diminution de la valence négative du cannabis peut expliquer la consommation élevée parmi les individus ayant un diagnostic de schizophrénie? Bien qu’un grand nombre de recherches pré-cliniques aient été menées chez l’animal normal pour évaluer l’effet motivationnel du Δ9-tétrahydrocannabinol (THC) et autres cannabinoïdes synthétiques, aucune n’a abordé cette problématique dans un modèle animal de la schizophrénie. Cette lacune nous a donc amené à étudier la valence motivationnelle du THC et de l’agoniste cannabinoïde WIN55,212-2 (WIN) dans un modèle animal de la schizophrénie: la lésion néonatale de l’hippocampe ventral (NVHL). Dans le premier article, nous présentons les résultats de quatre expériences. Une première avait pour objectif de déterminer si la procédure expérimentale que nous avons utilisée permettait de reproduire des signes distinctifs du modèle animal de la schizophrénie. Par la suite, nous avons évalué i) l’effet d’une dose de WIN sur l’activité locomotrice spontanée et ii) la valence motivationnelle du THC (0.5 mg/kg, i.p) et du WIN (1 mg/kg, i.p) chez les rats adolescents (jour post-natal 28-40, PD28-40) et adultes (PD56) au moyen du paradigme de préférence de place conditionnée (PPC). Tel qu’attendu, la réponse locomotrice à l’amphétamine (0.75 et 1.5 mg/kg) chez les rats NVHL adultes était supérieure à celle des rats contrôles (test distinctif du modèle). Le THC a induit une tendance aversive chez les rats contrôles adultes. Enfin, le WIN a stimulé l’activité locomotrice et induit une aversion significative chez les rats adultes NVHL. Dans un deuxième article, nous avons évalué la valence motivationnelle du THC (0.5 mg/kg), du WIN (1 et 3 mg/kg) et l’effet de l’amphétamine au moyen du paradigme d’autostimulation électrique intracérébrale (ASI). Les résultats montrent que : i) l’effet amplificateur de l’amphétamine sur l’ASI était de plus courte durée chez les rats NVHL; ii) le THC produit une légère atténuation de la récompense chez les rats contrôles tandis que le WIN a produit une atténuation plus prononcée de la récompense chez les rats NVHL, un effet qui a été bloqué par l’antagoniste aux récepteurs CB1, le AM251 (3 mg/kg). Pour la première fois les résultats suggèrent une altération du système endocannabinoïde dans un modèle animal de la schizophrénie. Ils indiquent qu’une exposition aigüe conduit à une prédominance de la valence négative. Bien qu’en apparente contradiction avec les études cliniques, ces résultats soulignent l’importance du contexte socio-environnemental pour expliquer les effets du cannabis chez les patients. De plus ils encouragent les futures études à évaluer cette valence sur un modèle d’exposition chronique.

Neurotensinergic modulation of glutamatergic neurotransmission in VTA neurons.

L’aire tegmentaire ventrale (VTA) contient une forte densité de terminaisons neurotensinergiques ainsi que des récepteurs à la surface des neurones dopaminergiques et non-dopaminergiques. Le VTA a été impliqué dans des maladies comme la schizophrénie, les psychoses et l’abus de substance. Les drogues d’abus sont connues pour induire le phénomène de sensibilisation - un processus de facilitation par lequel l’exposition à un stimulus produit une réponse augmentée lors de l’exposition subséquente au même stimulus. La sensibilisation se développe dans le VTA et implique mécanismes dopaminergiques et glutamatergiques. Il a été montré que les antagonistes neurotensinergiques bloquaient le développement de la sensibilisation et certains mécanismes de récompense et ces effets pourraient être médiés indirectement par une modulation de la neurotransmission glutamatergique. Cependant, on connaît peu les mécanismes de modulation de la transmission glutamatergique par la neurotensine (NT) dans le VTA. Le but de la présente thèse était d’étudier la modulation neurotensinergique de la neurotransmission glutamatergique dans les neurones dopaminergiques et non-dopaminergiques du VTA. Pour ce faire, nous avons utilisé la technique du patch clamp dans la cellule entière dans des tranches horizontales du VTA pour étudier les effets de différents agonistes et antagonistes neurotensinergiques. Les neurones ont été identifié comme Ih+ (présumés dopaminergiques) ou Ih- (présumés non-dopaminergiques) selon qu’ils exprimaient ou non un courant cationique activé par l’hyperpolarisation (Ih). Des techniques d’immunocytochimie ont été utilisées pour marquer les neurones et vérifier leur localisation dans le VTA. Dans une première étude nous avons trouvé que la neurotensine indigène (NT1-13) ou son fragment C-terminal, NT8-13, induisait une augmentation comparable des courants postsynaptiques excitateurs glutamatergiques (CPSEs) dans les neurones Ih+ ou Ih- du VTA. L'augmentation induite dans les neurones Ih+ par la NT8-13 a été bloquée par le SR48692, un antagoniste des récepteurs NTS1, et par le SR142948A, un antagoniste des récepteurs NTS1 et NTS2, suggérant que l'augmentation était médiée par l’activation des récepteurs NTS1. Dans les neurones Ih- l'augmentation n’a été bloquée que par le SR142948A indiquant une implication des récepteurs NTS2. Dans une deuxième étude, nous avons testé les effets de la D-Tyr[11]NT (un analogue neurotensinergique ayant différentes affinités de liaison pour les sous-types de récepteurs neurotensinergiques) sur les CPSEs glutamatergiques dans les neurones Ih+ et Ih- en parallèle avec une série d’expériences comportementales utilisant un paradigme de préférence de place conditionnée (PPC) menée dans le laboratoire de Pierre-Paul Rompré. Nous avons constaté que la D-Tyr[11]NT induisaient une inhibition dépendante de la dose dans les neurones Ih+ médiée par l'activation de récepteurs NTS2. En revanche, la D-Tyr[11]NT a produit une augmentation des CPSEs glutamatergiques médiée par des récepteurs NTS1 dans les neurones Ih-. Les résultats des expériences comportementales ont montré que des microinjections bilatérales de D-Tyr[11]NT dans le VTA induisait une PPC bloquée uniquement par la co-injection de SR142948A et SR48692, indiquant un rôle pour les deux types de récepteurs, NTS1 et NTS2. Cette étude nous a permis de conclure que i) la D-Tyr[11]NT agit dans le VTA via des récepteurs NTS1 et NTS2 pour induire un effet de récompense et ii) que cet effet est dû, au moins en partie, à une augmentation de la neurotransmission glutamatergique dans les neurones non-dopaminergiques (Ih-). Dans une troisième étude nous nous sommes intéressés aux effets de la D-Tyr[11]NT sur les réponses isolées médiées par les récepteurs N-méthyl-D-aspartate (NMDA) et acide α-amino-3- hydroxy-5-méthyl-4-isoxazolepropionique (AMPA) dans les neurones du VTA. Nous avons constaté que dans les neurones Ih+ l’amplitude des CPSEs NMDA et AMPA étaient atténuées de la même manière par la D-Tyr[11] NT. Cette modulation des réponses était médiée par les récepteurs NTS1 et NTS2. Au contraire, dans les neurones Ih-, l’amplitude des réponses NMDA et AMPA étaient augmentées en présence de D-Tyr[11]NT et ces effets dépendaient de l’activation des récepteurs NTS1 localisés sur les terminaisons glutamatergiques. Ces résultats fournissent une preuve supplémentaire que le NT exerce une modulation bidirectionnelle sur la neurotransmission glutamatergique dans les neurones du VTA et met en évidence un nouveau type de modulation peptidergique des neurones non-dopaminergiques qui pourrait être impliqué dans la sensibilisation. En conclusion, la modulation neurotensinergique de la neurotransmission glutamatergique dans les neurones dopaminergiques et non-dopaminergiques du VTA se fait en sens opposé soit, respectivement, par une inhibition ou par une excitation. De plus, ces effets sont médiés par différents types de récepteurs neurotensinergiques. En outre, nos études mettent en évidence une modulation peptidergique de la neurotransmission glutamatergique dans le VTA qui pourrait jouer un rôle important dans les mécanismes de lutte contre la toxicomanie.

Ethanol-induced sensitization depends preferentially on D(1) rather than D(2) dopamine receptors

Behavioral sensitization, defined as a progressive increase in the locomotor stimulant effects elicited by repeated exposure to drugs of abuse, has been used as an animal model for drug craving in humans. The mesoaccumbens dopaminergic system has been proposed to be critically involved in this phenomenon; however, few studies have been designed to systematically investigate the effects of dopaminergic antagonists on development and expression of behavioral sensitization to ethanol in Swiss mice. We first tested the effects of D(1) antagonist SCH-23390 (0-0.03 mg/kg) or D(2) antagonist Sulpiride (0-30 mg/kg) on the locomotor responses to an acute injection of ethanol (2.0 g/kg). Results showed that all tested doses of the antagonists were effective in blocking ethanol`s stimulant effects. In another set of experiments, mice were pretreated intraperitoneally with SCH-23390 (0.01 mg/kg) or Sulpiride (10 mg/kg) 30 min before saline or ethanol injection, for 21 days. Locomotor activity was measured weekly for 20 min. Four days following this pretreatment, all mice were challenged with ethanol. Both antagonists attenuated the development of ethanol sensitization, but only SCH-23390 blocked the expression of ethanol sensitization according to this protocol. When we tested a single dose (30 min before tests) of either antagonist in mice treated chronically with ethanol, both antagonists attenuated ethanol-induced effects. The present findings demonstrate that the concomitant administration of ethanol with D(1) but not D(2) antagonist prevented the expression of ethanol sensitization, suggesting that the neuroadaptations underlying ethanol behavioral sensitization depend preferentially on D(1) receptor actions. (C) 2010 Elsevier Inc. All rights reserved.