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Representation-mediated Aversion as a Model to Study Psychotic-like States in Mice
图示介导的厌恶作为模型研究小鼠精神病样状态   

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Abstract

Several paradigms for rodent models of the cognitive and negative endophenotypes found in schizophrenic patients have been proposed. However, significant efforts are needed in order to study the pathophysiology of schizophrenia-related positive symptoms. Recently, it has been shown that these positive symptoms can be studied in rats by using representation-mediated learning. This learning measure the accuracy of mental representations of reality, also called ‘reality testing’. Alterations in ‘reality testing’ performance can be an indication of an impairment in perception which is a clear hallmark of positive psychotic-like states. Thus, we describe here a mouse task adapted from previous findings based on a sensory preconditioning task. With this task, associations made between different neutral stimuli (e.g., an odor and a taste) and subsequent selective devaluation of one of these stimuli have allowed us to study mental sensory representations. Thus, the interest of this task is that it can be used to model positive psychotic-like states in mice, as recently described.

Keywords: Positive symptoms(阳性症状), Schizophrenia(精神分裂症), Delusions(妄想症), Reality testing(现实验证), Representation-mediated learning(图示介导的学习)

Background

The presence of positive symptoms, such as delusions or hallucinations, is a key feature of a psychotic-like state (American Psychiatric Association, 2000 and 2013; Tandon, 2013) and represents a major challenge to rodent models (Wong and Van Tol, 2003; van den Buuse et al., 2005; Mouri et al., 2007; Jones et al., 2011). Indeed, these symptoms are often neglected due to the lack of suitable animal models (Jones et al., 2011; Rubino and Parolaro, 2014 and 2016). Psychotogenic drug-induced hyperlocomotion in rodents has long been considered an acceptable approximation of positive symptoms of drug-induced psychotic-like states (Wong and Van Tol, 2003; van den Buuse et al., 2005; Mouri et al., 2007; Jones et al., 2011). However, locomotor activity cannot be used to study the mismatch between perception and reality that is the hallmark of positive psychotic-like states (Wong and Van Tol, 2003; van den Buuse et al., 2005). For instance, the ‘Diagnostic and Statistical Manual of Mental Disorders’ (DSM V) defines delusions as ‘erroneous beliefs that usually involve a misinterpretation of perception or experiences’ (American Psychiatric Association, 2013). To overcome this methodological limitation, recent studies have used behavioral procedures in rodents designed to measure the accuracy of mental representations of reality, also called ‘reality testing’ (McDannald and Schoenbaum, 2009; McDannald et al., 2011; Kim and Koh, 2016). Alterations in ‘reality testing’ performance can be an indication of an impairment in perception which, as we mentioned before, might lead to positive psychotic-like states such as delusions. Thus, we adapt these previous protocols (McDannald and Schoenbaum, 2009; McDannald et al., 2011; Wheeler et al., 2013; Kim and Koh, 2016) to design a behavioral paradigm for measuring ‘reality testing’ in mice (Busquets-Garcia et al., 2017). Notably, deficits in task performance induced by psychotogenic drugs (e.g., amphetamine or cannabinoids) seem to reflect the kind of perceptual alterations that are the hallmarks and early features of positive psychotic-like symptoms.

Materials and Reagents

  1. 1 ml syringe and 26 G needles (Terumo Europe, Leuven, Belgium)
  2. Mice (C57BL6/N mice purchased from Janvier Labs, Le Genest-Saint-Isle, France)
  3. Banana odor (isoamyl acetate) (Sigma-Aldrich, catalog number: W205508 )
  4. Almond odor (benzaldehyde) (Sigma-Aldrich, catalog number: 418099 )
  5. Maltodextrin (Sigma-Aldrich, catalog number: 419672 )
  6. Sucrose (Sigma-Aldrich, catalog number: S0389 )
  7. Lithium chloride (LiCl) (Sigma-Aldrich, catalog number: 203637 )
  8. Banana solution (0.05%) (see Recipes)
  9. Almond solution (0.01%) (see Recipes)
  10. Maltodextrin solution (5%) (see Recipes)
  11. Sucrose solution (5%) (see Recipes)
  12. Combined odor-taste solution (see Recipes)
  13. LiCl (3 M) (see Recipes)

Note: Any psychotogenic drug (e.g., amphetamine or cannabinoids) can be used as a positive control for the test, as previously shown (Busquets-Garcia et al., 2017).

Equipment

  1. 50-ml drinking tubes (Conical Centrifuge Tube, Thermo Fisher Scientific, Rochester, USA)
  2. Standard balance
  3. Standard individual plexiglas cage for each mouse
  4. Metal tube (TD-100, 2.5” straight ball point tube, stainless steel, Ancare, New York, USA)
  5. Rubber plug with 25 mm diameter hole (Thermo Fisher Scientific, Rochester, USA)

Note: It is important that the bottles be assembled before starting the experiment (Figure 1).


Figure 1. Drinking bottles used in the protocol. The bottles are carefully put together, as shown in the image. Note that the metal tube must pass through the rubber plug and all possible leakage must be avoided.

Procedure

  1. Place mice in individual cages in a room with controlled temperature (21 ± 1 °C) and humidity (55 ± 10%).
  2. Mice are water deprived 24 h before the first day of the protocol. This water deprivation lasts for the full duration of the protocol and mice weight is also controlled (maintained at around 85-90% of original).
  3. All animals receive 1-h access to water (using the drinking bottles, Figure 1). The bottle is weighed before and after this period in order to precisely quantify how much the animals drink in each session. This procedure is repeated during three consecutive days as a habituation period.
    Notes:
    1. If a mouse does not drink more than 1 ml in one hour, it is given access to the bottle for one extra hour. This is particularly important the first day when animals are not yet habituated.
    2. The position of the bottle is changed every day (right or left, see Figure 2). The height and angle of the bottles is maintained throughout the protocol.


      Figure 2. Position of the bottles. The position of the bottles is changed during the protocol (see Procedure) in order to habituate the mice to drink from both positions. However, bottles must be maintained at the same height and angle throughout the protocol. Note that food is distributed in both sides of the metal grid.

  4. Over the following days, conduct the odor-taste pairing phase (preconditioning, Figure 3) in which animals are exposed to 6 odor-taste pairings. Each pairing consists of two days: the first day, animals are given 1-h access to a flavored water-based solution containing a novel taste (either 5% sucrose or 5% maltodextrin; Taste 1, T1) and a novel odorant (0.05 % Banana or 0.01 % Almond; Odor 1, O1) in order to pair T1 with O1. On day two, animals are given access to a new taste and odor, neither of which has been presented the previous day, i.e., Taste 2 (T2) and Odor 2 (O2).
    Notes:
    1. Any new mouse strain or genetic mouse model must first be tested using 3 odor-taste pairings (instead of 6) in order to establish whether the mouse line in question is apt to present mediated aversion (Busquets-Garcia et al., 2017). In our recent work, control mice (e.g., C57BL6/N) present clear mediated aversion after 3 pairings. However, when the number of training sessions during preconditioning is increased (6 or 9 pairings), animals no longer present mediated aversion. For this reason, we used the 6 pairing protocol to study the acute effects of psychotogenic drugs (Busquets-Garcia et al., 2017). However, when using a new line (e.g., CB1 knockout mice), it must first be established that the animals are able to show mediated aversion with limited training before they are tested in long training protocols. It is likely that certain mouse models will present impaired development of mediated aversion. Indeed, odor-mediated taste learning has long been used as a learning test wherein different genotypes or mouse models present characteristic performances in mediated aversion.
    2. The position of the bottles is changed for each pairing (right or left, see Figure 2).
    3. The preference for specific odors or tastes has been demonstrated recently (Busquets-Garcia et al., 2017).
    4. The odor-taste solutions are prepared anew every 2-3 days and all bottles are thoroughly cleaned at the end of the preconditioning phase to avoid any residual taste persisting into the following session.


    Figure 3. Simple representation of the behavioural paradigm. Schema of the protocol used to assess odor-mediated taste aversion (top) and taste-mediated odor aversion (bottom). For a more detailed schema refer to Busquets-Garcia et al., 2017. Before preconditioning, all animals receive 1-h access to water during three consecutive days as water habituation. Next, conduct the odor-taste pairings phase, which consists of 6 odor-taste pairings (Busquets-Garcia et al., 2017). Each pairing lasts two days: the first day, animals receive 1-h access to a flavored solution containing a novel taste (T1) and a novel odorant (O1) in order to pair T1 with O1. On day two, animals receive whichever taste and odor have not been presented the previous day, i.e., T2 and O2. On the following days, animals enter the conditioning (or devaluation) phase where either O1 (top) or T1 (bottom) is devaluated. On days 1, 3 and 5 of this phase, animals receive 1-h access to O2 (top) or T2 (bottom) immediately followed by an intraperitoneal (i.p.) injection of saline, whereas on days 2, 4 and 6, they receive 1-h access to O1 (top) or T1 (bottom) immediately followed by an i.p. injection of the visceral malaise-inducing drug lithium chloride (LiCl, 0.3 M, 1% b.w.). After this conditioning, animals are given one day for recovery in which they receive water during 1 h. On the next 2 days, mediated and direct aversions are assessed using a 1-h two-choice test (top and bottom). Mediated aversion is evaluated in Test 1 by means of a choice between the mediated CS+ (mCS+: consisting of the taste [top] or odor [bottom] stimulus which has been previously associated with the LiCl-paired CS odor or taste, respectively) and the mediated CS- (mCS-: consisting of the taste [top] or odor [bottom] stimulus which has been previously associated with the saline-paired CS odor or taste, respectively). If, on the same day, researchers are interested to know the effect of a given drug on the mediated aversion test (Busquets-Garcia et al., 2017), acute treatments can be performed just before the test. Then, on the second day, direct aversion is evaluated by means of a choice between the conditioned LiCl-paired stimulus (CS+) and the conditioned saline-paired stimulus (CS-).

  5. During the following 6 days, animals undergo the devaluation phase (Figure 3) whereby O1 is devaluated to become the conditioned stimulus (CS+). On days 1, 3 and 5 of this phase, animals receive 1-h access to O2 followed by an intraperitoneal (i.p.) injection of saline (CS-), whereas on days 2, 4 and 6, they receive 1-h access to O1 immediately followed by an i.p. injection of the emetic agent LiCl (0.3 M, 1% b.w. i.e., 10 ml/kg) (CS+) in order to induce gastric malaise.
    Notes:
    1. To assess taste-mediated odor aversion, the devaluation phase pairing is performed using the tastes. T2 is associated with a saline injection (CS-) whereas T1 is associated with a LiCl injection (CS+).
    2. The position of the bottles is changed for each pairing (right or left, see Figure 2). The height and angle of the bottles is maintained throughout the protocol.
    3. The LiCl injection is performed just after removal of the bottles. A few minutes after injection, animals are observed to experience dizziness and greatly reduced locomotion.
    4. Odor or taste solutions are prepared anew every 2-3 days and all bottles are thoroughly cleaned at the end of this phase.
  6. Animals are given one day for recovery in which they receive water for 1 h.
    Note: The position of the water bottle is opposite that of the last pairing. 
  7. On the next day, mediated aversion is assessed using a 1-h two-choice test where two different bottles are presented at the same time (Figure 4). Mediated aversion is always evaluated on the first day of tests with a choice between the mCS+ and the mCS-. The placement of each bottle is counterbalanced to avoid any position bias in task performance. From our experience, control animals with intermediate training (3 pairings) during preconditioning develop mediated aversion. Notably, if the training sessions are increased (6 or 9 pairings), the animals do not present mediated aversion (see Busquets-Garcia et al., 2017 for further information).
    Notes:
    1. To assess taste-mediated odor aversion, this two-choice test is carried out using the two different odor bottles.
    2. At the beginning of this test session, a ‘forced sampling’ procedure is used to ensure that the mice sample both fluids. Both bottles are presented: when the mouse has licked one of the bottles, this bottle is removed until the mouse licks the second bottle. When this has happened, the second bottle is also removed. Finally, both bottles are then presented simultaneously. The order of presentation of the bottles during forced sampling is counterbalanced to avoid any order bias. 


    Figure 4. Two-choice test. During the two-choice test two bottles (i.e., tastes or odors) are made available for duration of one hour. In order to make the animal aware of the choice, perform a ’forced sampling’ procedure following these steps: (i) both bottles are presented at the same time, (ii) when the animal drink from one bottle this bottle is removed, (iii) time is allowed for the mouse to drink from the remaining bottle, (iv) the second bottle is removed and, finally, (v) both bottles are re-introduced simultaneously, as seen in this image, in order for the two-choice test to be performed.

  8. On the second day, direct aversion is evaluated using a two-choice test between the CS+ and CS- with a procedure identical to that described for the mediated aversion test.

Data analysis

Aversion was revealed by lower consumption of mCS+ than mCS- (mediated aversion) or of CS+ than CS- (direct aversion) (Soria-Gomez et al., 2015; Busquets-Garcia et al., 2017). Data were presented as absolute liquid intake and an aversion index was calculated using the following formula: (CS- - CS+)/(CS+ + CS-) for direct aversion or (mCS- - mCS+)/(mCS+ + mCS-) for mediated aversion, respectively.

Notes

  1. All animals were housed in individual cages for at least 5 days before the experiment.
  2. All sessions of the experimental protocol were performed in the morning (from 9 AM to 1 PM).
  3. In order to avoid any olfactory contamination between bottles, 3 different bottles were used: some bottles were exclusively used for banana odor or the mixture of banana odor with a taste, either sucrose or maltodextrin (banana bottles), others only for almond odors or the mixture of almond odor with a taste (almond bottles) and the remaining bottles were used for water or taste alone (water/taste bottles). Bedding was changed at the start of the protocol and after the preconditioning phase, some hours after the last session.
  4. The task was tested in several mice lines. We found similar results between the C57/BL6N mice and the wild-type animals produced in our institute.

Recipes

  1. Banana solution (0.05%)
    To each 100 ml of water, we added 50 μl of isoamyl acetate. In order to prepare this solution, we put the necessary quantity of water in a container, to which we added the previously calculated quantity of odor. The solution was then mixed until the odor dissolved
  2. Almond solution (0.01%)
    To each 100 ml of water, we added 10 μl of benzaldehyde. In order to prepare this solution, we put the necessary quantity of water in a container, to which we added the previously calculated quantity of odor. The solution was then mixed until the odor dissolved
  3. Maltodextrin solution (5%)
    To each 100 ml of water, we added 5 g of maltodextrin. In order to prepare this solution, we put the necessary quantity of water in a container, to which we added the previously calculated quantity of taste. The solution was then mixed until the powder dissolved
  4. Sucrose solution (5%)
    To each 100 ml of water, we added 5 g of sucrose. In order to prepare this solution, we put the necessary quantity of water in a container, to which we added the previously calculated quantity of taste. The solution was then mixed until the powder dissolved
  5. Combined odor-taste solution
    Following the same quantities as above, we weighed out the required quantity of sucrose or maltodextrin, to which we added the necessary quantity of water and, finally, odor. The solution was then thoroughly mixed until full dissolution and transparency had been achieved
  6. LiCl (3 M)
    We weighed out 0.627 g of the LiCl powder and added it to 50 ml of physiological saline. The solution was then mixed thoroughly until the powder dissolved

Acknowledgments

We thank Delphine Gonzales, Nathalie Aubailly, and all the personnel of the Animal Facility of the NeuroCentre Magendie for mouse care. We would also like to thank all those who have performed experimental work using this task: Yarmo Mackenbach, Bastien Redon, Carolina Muguruza, Geoffrey Terral, Camile Pernegre, Christina Ioannidou and Marjorie Varilh. We also thank Christopher Stevens for the critical reading of the manuscript. This protocol was adapted according to McDannald MA et al. (2011). This work was supported by INSERM (to G.M.), INRA (G.F.), EU-FP7 (PAINCAGE, HEALTH-603191 to G.M. and FP7-PEOPLE-2013-IEF-623638 to A.B.-G.), European Research Council (Endofood, ERC-2010-StG-260515; CannaPreg, ERC-2014-PoC-640923, to G.M.), Fondation pour la Recherche Medicale (DRM20101220445 and DPP20151033974, to G.M.), Human Frontiers Science Program (to G.M.), Region Aquitaine (to G.M.), French State/Agence Nationale de la Recherche (BRAIN ANR-10-LABX-0043 to G.M., ANR-10-IDEX-03- 02 to A.B.-G, NeuroNutriSens ANR-13-BSV4-0006-02 to G.M. and ORUPS ANR-16-CE37-0010 to G.M. and G.F.), Fyssen Foundation (to E.S.-G.) and CONACyT (to E.S.-G.).

References

  1. American Psychiatric Association. (2000). DSM, 4th edition. Washington, DC: APS.
  2. American Psychiatric Association. (2013). DSM, 5th edition. Washington, DC: APS.
  3. Busquets-Garcia, A., Soria-Gomez, E., Redon, B., Mackenbach, Y., Vallee, M., Chaouloff, F., Varilh, M., Ferreira, G., Piazza, P. V. and Marsicano, G. (2017). Pregnenolone blocks cannabinoid-induced acute psychotic-like states in mice. Mol Psychiatry.
  4. Jones, C. A., Watson, D. J. and Fone, K. C. (2011). Animal models of schizophrenia. Br J Pharmacol 164(4): 1162-1194.
  5. Kim, H. J. and Koh, H. Y. (2016). Impaired reality testing in mice lacking phospholipase Cβ1: observed by persistent representation-mediated taste aversion. PLoS One 11(1): e0146376.
  6. McDannald, M. A., Whitt, J. P., Calhoon, G. G., Piantadosi, P. T., Karlsson, R. M., O'Donnell, P. and Schoenbaum, G. (2011). Impaired reality testing in an animal model of schizophrenia. Biol Psychiatry 70(12): 1122-1126.
  7. McDannald, M. and Schoenbaum, G. (2009). Toward a model of impaired reality testing in rats. Schizophr Bull 35(4): 664-667.
  8. Mouri, A., Noda, Y., Enomoto, T. and Nabeshima, T. (2007). Phencyclidine animal models of schizophrenia: approaches from abnormality of glutamatergic neurotransmission and neurodevelopment. Neurochem Int 51(2-4): 173-184.
  9. Rubino, T. and Parolaro, D. (2014). Cannabis abuse in adolescence and the risk of psychosis: a brief review of the preclinical evidence. Prog Neuropsychopharmacol Biol Psychiatry 52: 41-44.
  10. Rubino, T. and Parolaro, D. (2016). The impact of exposure to cannabinoids in adolescence: insights from animal models. Biol Psychiatry 79(7): 578-585.
  11. Soria-Gomez, E., Busquets-Garcia, A., Hu, F., Mehidi, A., Cannich, A., Roux, L., Louit, I., Alonso, L., Wiesner, T., Georges, F., Verrier, D., Vincent, P., Ferreira, G., Luo, M. and Marsicano, G. (2015). Habenular CB1 receptors control the expression of aversive memories. Neuron 88(2): 306-313.
  12. Tandon, R. (2013). Definition of psychotic disorders in the DSM-5 too radical, too conservative, or just right! Schizophr Res 150(1): 1-2.
  13. van den Buuse, M., Garner, B., Gogos, A. and Kusljic, S. (2005). Importance of animal models in schizophrenia research. Aust N Z J Psychiatry 39(7): 550-557.
  14. Wheeler, D. S., Chang, S. E. and Holland, P. C. (2013). Odor-mediated taste learning requires dorsal hippocampus, but not basolateral amygdala activity. Neurobiol Learn Mem 101: 1-7.
  15. Wong, A. H. and Van Tol, H. H. (2003). Schizophrenia: from phenomenology to neurobiology. Neurosci Biobehav Rev 27(3): 269-306.

简介

已经提出了在精神分裂症患者中发现的认知和负面内型的啮齿动物模型的几个范例。然而,为了研究精神分裂症相关阳性症状的病理生理学,需要作出重大努力。最近已经表明,通过使用代表介导的学习,可以在大鼠中研究这些阳性症状。这种学习测量了现实心理表征的准确性,也称为“现实测试”。 “现实测试”表现的改变可以表明感知障碍,这是积极的精神病样状态的明显标志。因此,我们在这里介绍一种基于感官预处理任务的以前发现的改编的鼠标任务。通过这项任务,在不同的中性刺激(例如气味和味道)之间进行的协会以及随后的这些刺激之一的选择性贬值使我们能够研究精神感觉表征。因此,这个任务的兴趣在于,它可以用于模拟小鼠中的阳性类似精神状态,如最近所描述的。
【背景】阳性症状(如妄想或幻觉)的存在是精神病样状态的关键特征(美国精神病学协会,2000年和2013年; Tandon,2013年),并且是啮齿动物模型的重大挑战(Wong and Van Tol,2003 ; van den Buuse等人,2005; Mouri等人,2007; Jones等人,2011)。实际上,由于缺乏合适的动物模型(Jones et al。,2011; Rubino和Parolaro,2014和2016),这些症状往往被忽略。长期以来,啮齿动物中的精神病药物诱导的超运动长期以来被认为是药物诱发的精神病样状态的阳性症状的可接受的近似值(Wong和Van Tol,2003; van den Buuse et al。,2005; Mouri et al。,2007; Jones et al。,2011)。然而,运动活动不能用于研究作为阳性精神病样状态的标志的感知与现实之间的不匹配(Wong和Van Tol,2003; van den Buuse et al。,2005)。例如,“精神障碍诊断和统计手册”(DSM V)将妄想定义为“通常涉及感知或经验误解的错误信念”(美国精神病学协会,2013年)。为了克服这种方法学上的限制,最近的研究已经使用了设计用于测量现实心理表征精确度的啮齿动物的行为程序(也称为“现实测试”)(McDannald和Schoenbaum,2009; McDannald等,2011; Kim and Koh,2016; )。 “现实测试”表现的改变可以表明感知障碍,如我们前面提到的那样,可能导致像精神病这样的类似状态的妄想。因此,我们适应这些以前的方案(McDannald和Schoenbaum,2009; McDannald等,2011; Wheeler等,2013; Kim和Koh,2016)设计了一种用于测量小鼠“现实测试”的行为模式(Busquets- Garcia et al。,2017)。值得注意的是,由精神病药物(例如安非他明或大麻素)引起的任务绩效的赤字似乎反映了作为阳性精神病样症状的特征和早期特征的感知改变的种类。

关键字:阳性症状, 精神分裂症, 妄想症, 现实验证, 图示介导的学习

材料和试剂

  1. 1 ml注射器和26 G针(Terumo Europe,Leuven,Belgium)
  2. 小鼠(从Janvier Labs,Le Genest-Saint-Isle,France购买的C57BL6/N小鼠)
  3. 香蕉气味(乙酸异戊酯)(Sigma-Aldrich,目录号:W205508)
  4. 杏仁味(苯甲醛)(Sigma-Aldrich,目录号:418099)
  5. 麦芽糖糊精(Sigma-Aldrich,目录号:419672)
  6. 蔗糖(Sigma-Aldrich,目录号:S0389)
  7. 氯化锂(LiCl)(Sigma-Aldrich,目录号:203637)
  8. 香蕉溶液(0.05%)(见配方)
  9. 杏仁溶液(0.01%)(见配方)
  10. 麦芽糊精溶液(5%)(参见食谱)
  11. 蔗糖溶液(5%)(参见食谱)
  12. 合并的气味味道溶液(见食谱)
  13. LiCl(3 M)(见配方)

注意:如先前所示,任何精神病药(例如苯丙胺或大麻素)可用作测试的阳性对照(Busquets-Garcia等,2017)。

设备

  1. 50 ml饮水管(Conical Centrifuge Tube,Thermo Fisher Scientific,Rochester,USA)
  2. 标准平衡
  3. 标准个别有机玻璃笼为每只老鼠
  4. 金属管(TD-100,2.5"直球点管,不锈钢,Ancare,纽约,美国)
  5. 25 mm直径孔的橡胶塞(Thermo Fisher Scientific,Rochester,USA)

注意:在开始实验之前,必须组装瓶子(图1)。


图1.协议中使用的饮用瓶。如图所示,将瓶子小心地放在一起。请注意,金属管必须穿过橡胶塞,并且必须避免所有可能的泄漏。

程序

  1. 将小鼠放在具有控制温度(21±1℃)和湿度(55±10%)的房间中的单个笼中。
  2. 小鼠在方案第一天之前24小时被水剥夺。这种水分剥夺持续了协议的全部时间,小鼠的体重也受到控制(保持在原始的85-90%左右)。
  3. 所有动物接受水1小时(使用饮用水,图1)。在此期间之前和之后,将瓶子称重,以精确量化每次饮食中多少动物。这个程序在连续三天作为习惯期重复。
    注意:
    1. 如果一只小鼠在一小时内不能喝1毫升以上的饮料,则可以使用瓶子一个多小时。这在动物尚未习惯的第一天尤其重要。
    2. 每天更换瓶子的位置(右或左,见图2)。在整个协议中保持瓶子的高度和角度。


      图2.瓶子的位置。瓶子的位置在方案中改变(参见程序),以便习惯从两个位置喝小鼠。然而,瓶子必须在整个协议中保持在相同的高度和角度。请注意,食物分布在金属网格的两侧。

  4. 在接下来的日子里,进行气味 - 配对阶段(预处理,图3),其中动物暴露于6种气味味配对。每个配对由两天组成:第一天,动物接受含有新味道(5%蔗糖或5%麦芽糖糊精;味道1,T1)和新颖加味剂(0.05)的口味水溶液1小时%香蕉或0.01%杏仁;气味1,O1),以便将T1与O1配对。在第二天,动物可以获得新的味道和气味,这些都不是前一天提出的,例如,味道2(T2)和气味2(O2)。 > 注意:
    1. 必须首先使用3种气味 - 配对(而不是6种)来测试任何新的小鼠品系或遗传小鼠模型,以便确定所讨论的小鼠品系是否易于呈现介导的厌恶(Busquets-Garcia等人, 2017年)。在我们最近的工作中,对照小鼠(例如,C57BL6/N)在3次配对后呈现明显的介导的厌恶。然而,当预处理期间的训练次数增加(6或9对)时,动物不再存在介导厌恶。因此,我们使用6配对方案来研究精神病药物的急性影响(Busquets-Garcia et al。,2017)。然而,当使用新的线(例如,CB1敲除小鼠)时,必须首先确定动物能够在有限的训练之前显示介导的厌恶,然后才能在长训练方案中进行测试。某些小鼠模型很可能会导致中介厌恶的发展受损。事实上,气味介导的味觉学习长期以来一直用作学习测试,其中不同的基因型或小鼠模型在介导厌恶中呈现特征性表现。

    2. 最近已经证明了对特定气味或口味的偏好(Busquets-Garcia et al。,2017)。
    3. 气味味道溶液每2-3天重新制备,所有瓶子在预处理阶段结束时彻底清洁,以避免残留味道持续到下一个阶段。


    图3.行为范例的简单表示用于评估气味介导的味觉厌恶(上)和味道介导的气味厌恶(底部)的方案的模式。对于更详细的模式,请参阅布加斯加 - 加西亚等人,2017年。在预处理之前,所有动物在连续三天内获得1小时的水供水习惯。接下来,进行气味味配对阶段,其由6种气味配对(Busquets-Garcia等人,2017)组成。每次配对持续两天:第一天,动物接受1小时的通路,可以获得含有新味道(T1)和新颖的气味剂(O1)的风味溶液,以便将T1与O1配对。在第二天,动物收到前一天没有出现的味道和气味,即,T2和O2。在接下来的日子里,动物进入O1(上)或T1(底部)贬值的调理(或贬值)阶段。在这个阶段的第1,3和5天,动物接受1小时的通气,接近O2(上)或T2(底部),然后腹膜内(ip)注射盐水,而在第2,4和6天,他们接受1-h访问O1(上)或T1(底部)紧随其后的ip注射内脏不适诱导药物氯化锂(LiCl,0.3M,1%b.w.)。在这种调理之后,给动物一天进行恢复,在1小时内他们接受水。在接下来的2天中,使用1小时双向选择测试(顶部和底部)评估介导和直接反应。在试验1中通过介导的CS +(mCS +:分别由以前与LiCl-配对的CS气味或味道相关联的味道[顶部]或气味[底部]刺激组成)之间的选择来评估介导的厌恶)和介导的CS-(mCS-:分别由先前与盐水对应的CS气味或味道相关联的味道[顶部]或气味[底部]刺激组成)。如果在同一天,研究人员有兴趣了解给定药物对介导的厌恶测试(Busquets-Garcia等人,2017年)的影响,则可以在测试之前进行急性治疗。然后,在第二天,通过调节的LiCl配对刺激(CS +)和条件生理盐水配对刺激(CS-)之间的选择来评估直接厌恶。

  5. 在接下来的6天内,动物经历贬值阶段(图3),由此O1贬值成为条件刺激(CS +)。在该阶段的第1,3和5天,动物接受1小时的通气,然后腹膜内(ip)注射盐水(CS-),而在第2,4和6天,他们接受1小时的通路O1紧随其后注射催吐剂LiCl(0.3M,1%b.w.,即10ml/kg)(CS +)以诱导胃部不适。
    注意:
    1. 为了评估味觉介导的气味厌恶,使用口味进行贬值阶段配对。 T2与盐水注射(CS-)相关,而T1与LiCl注射(CS +)相关。
    2. 每个配对都会更改瓶子的位置(右侧或左侧见图2)。在整个协议中保持瓶的高度和角度。
    3. 在取出瓶子之后执行LiCl注射。注射后几分钟,动物观察到头晕,大大减轻了运动。
    4. 气味或味道溶液每2-3天重新制备一次,所有瓶子在本阶段结束时彻底清洗。
  6. 动物被给予一天的恢复,在那里他们接受水1小时。
    注意:水瓶的位置与最后配对的位置相反。 
  7. 在第二天,使用1小时双选择测试来评估介导厌恶,其中两个不同的瓶子同时呈现(图4)。介入厌恶总是在测试的第一天进行评估,并选择mCS +和mCS-。每个瓶子的放置是平衡的,以避免任务性能中的任何位置偏差。根据我们的经验,在预处理过程中控制中等训练的动物(3对)发展中介厌恶。值得注意的是,如果培训课程增加(6对或9对),动物不会出现中介厌恶(参见Busquets-Garcia et al。,2017,以获取更多信息)。
    注意:
    1. 为了评估味觉介导的气味厌恶,这种双选择测试使用两种不同的气味瓶进行。
    2. 在本次测试开始时,使用"强制取样"程序来确保小鼠对两种流体进行采样。两个瓶子都呈现:当鼠标舔了一个瓶子之后,这个瓶子被取出,直到舔第二个瓶子。当发生这种情况时,第二瓶也被移除。最后,两瓶同时呈现。在强制抽样过程中瓶子的展示顺序是平衡的,以避免任何订单偏差。 


    图4.双选项测试在双选项测试期间,两个瓶子( ,品味或气味)在一小时内可用。为了使动物意识到选择,请按照以下步骤执行"强制取样"程序:(i)两个瓶子同时呈现,(ii)当从一瓶瓶中取出动物饮料时, iii)允许老鼠从剩余的瓶子中饮用时间,(iv)移除第二瓶,最后,(v)两个瓶子同时重新引入,如图所示,选择测试执行。

  8. 在第二天,使用CS +和CS-之间的两选择测试来评估直接厌恶,其过程与介绍的厌恶测试所述相同。

数据分析

厌恶是通过mCS +的消耗低于mCS-(介导的厌恶)或CS +比CS-(直接厌恶)(Soria-Gomez等人,2015年; Busquets-Garcia等) 。,2017)。将数据作为绝对液体摄入量,使用以下公式计算厌恶指数:用于直接厌恶或(mCS- - mCS +)/(mCS + + mCS-)的介导的(CS- - CS +)/(CS + + CS-)厌恶。

笔记

  1. 所有动物在实验前至少保存5天
  2. 实验方案的所有会议在早晨(上午9点至下午1点)进行。
  3. 为了避免瓶子之间发生任何嗅觉污染,使用了3种不同的瓶子:一些瓶子专门用于香蕉气味或香味香气混合物,即蔗糖或麦芽糖糊精(香蕉瓶),其他仅适用于杏仁气味或混合杏仁味与味道(杏仁瓶)和剩余的瓶子单独使用水或味道(水/味道瓶)。床上用品在协议开始时和预处理阶段之后,在上一届会议后几个小时更改
  4. 该任务在几条小鼠线上进行了测试。我们在C57/BL6N小鼠和我们研究所生产的野生型动物之间发现了类似的结果

食谱

  1. 香蕉溶液(0.05%)
    向每100ml水中加入50μl乙酸异戊酯。为了准备这个解决方案,我们把必要数量的水放在一个容器中,我们添加了以前计算出的气味量。然后将溶液混合直至气味溶解
  2. 杏仁溶液(0.01%)
    向每100ml水中加入10μl苯甲醛。为了准备这个解决方案,我们把必要数量的水放在一个容器中,我们添加了以前计算出的气味量。然后将溶液混合直至气味溶解
  3. 麦芽糊精溶液(5%)
    向每100ml水中加入5g麦芽糖糊精。为了准备这个解决方案,我们把必要数量的水放在一个容器中,我们添加了以前计算出的味道数量。然后将溶液混合直至粉末溶解为
  4. 蔗糖溶液(5%)
    向每100ml水中加入5g蔗糖。为了准备这个解决方案,我们把必要数量的水放在一个容器中,我们添加了以前计算出的味道数量。然后将溶液混合直至粉末溶解为
  5. 组合气味味溶液
    按照与上述相同的数量,我们称量所需量的蔗糖或麦芽糖糊精,我们添加了必要量的水,最后是气味。然后将溶液充分混合,直到完全溶解,透明度达到
  6. LiCl(3 M)
    称量0.627g LiCl粉末并加入到50ml生理盐水中。然后将溶液充分混合直至粉末溶解为

致谢

我们感谢Delphine Gonzales,Nathalie Aubailly以及NeuroCentre Magendie动物设施的所有人员进行鼠标护理。我们还要感谢所有执行实验工作的人:Yarmo Mackenbach,Bastien Redon,Carolina Muguruza,Geoffrey Terral,Camile Pernegre,Christina Ioannidou和Marjorie Varilh。我们还感谢Christopher Stevens对稿件的批判性阅读。该方案根据McDannald MA等人进行了修改。 (2011年)。这项工作得到了INSERM(对GM),INRA(GF),EU-FP7(PAINCAGE,HEALTH-603191至GM和FP7-PEOPLE-2013-IEF-623638至AB-G)的支持,欧洲研究委员会(Endofood, ERC-2010-StG-260515; CannaPreg,ERC-2014-PoC-640923,转基因),基金会基金会(DRM20101220445和DPP20151033974,GM),人类前沿科学计划(GM),阿基坦地区),法国国家/国民大会(GLAIN ANR-10-LABX-0043至GM,ANR-10-IDEX-03- 02至AB-G,NeuroNutriSens ANR-13-BSV4-0006-02至GM和ORUPS ANR-16-CE37-0010给GM和GF),Fyssen基金会(ES-G。)和CONACyT(到ES-G。)。

参考

  1. 美国精神病学协会。 (2000)。< a class ="ke-insertfile"href ="http://dsm.psychiatryonline.org/doi/abs/10.1176/appi.books.9780890420249.dsm-iv-tr"target ="_ blank "> DSM,第4版。华盛顿特区:APS。
  2. 美国精神病学协会。 (2013)。帝斯曼,第5版。 ,DC:APS。
  3. Busquets-Garcia,A.,Soria-Gomez,E.,Redon,B.,Mackenbach,Y.,Vallee,M.,Chaouloff,F.,Varilh,M.,Ferreira,G.,Piazza,PV and Marsicano, G.(2017)。孕烯醇酮阻断大麻素引起的急性精神病样状态在小鼠中。 Mol Psychiatry
  4. Jones,CA,Watson,DJ and Fone,KC(2011)。  精神分裂症的动物模型。 Br J Pharmacol 164(4):1162-1194。
  5. Kim,HJ和Koh,HY(2016)。受损在缺乏磷脂酶Cβ1的小鼠中的现实测试:通过持续表达介导的味觉厌恶观察到。 11(1):e0146376。
  6. McDannald,MA,Whitt,JP,Calhoon,GG,Piantadosi,PT,Karlsson,RM,O'Donnell,P.和Schoenbaum,G。(2011)。精神分裂症动物模型中的受损现实测试生物精神病学 70(12) :1122-1126。
  7. McDannald,M.和Schoenbaum,G。(2009)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/19460880"target ="_ blank" >向大鼠受损现实测试的模型。
    Schizophr Bull 35(4):664-667。
  8. Mouri,A.,Noda,Y.,Enomoto,T。和Nabeshima,T。(2007)。精神分裂症苯丙利啶动物模型:谷氨酸能神经传递和神经发育异常的方法。神经化学Int 51(2-4):173-184。
  9. Rubino,T.和Parolaro,D。(2014)。青少年大麻滥用和精神病的风险:临床前证据的简要回顾。Prog Neuropsychopharmacol Biol Psychiatry 52:41-44。
  10. Rubino,T.和Parolaro,D。(2016)。暴露于大麻素在青春期的影响:动物模型的见解生物精神病学杂志 79(7):578-585。
  11. Soria-Gomez,E.,Busquets-Garcia,A.,Hu,F.,Mehidi,A.,Cannich,A.,Roux,L.,Louit,I.,Alonso,L.,Wiesner,T.,Georges ,F.,Verrier,D.,Vincent,P.,Ferreira,G.,Luo,M。和Marsicano,G。(2015)。 Habenular CB <1> 受体控制厌恶记忆的表达。神经元 88(2):306-313。
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引用:Busquets-Garcia, A., Soria-Gómez, E., Ferreira, G. and Marsicano, G. (2017). Representation-mediated Aversion as a Model to Study Psychotic-like States in Mice. Bio-protocol 7(12): e2358. DOI: 10.21769/BioProtoc.2358.
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