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Touchscreen-based Visual Discrimination and Reversal Tasks for Mice to Test Cognitive Flexibility
采用基于触摸屏的小鼠视觉辨别和颠倒任务测试认知灵活性   

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参见作者原研究论文

本实验方案简略版
Frontiers in Behavioral Neuroscience
Mar 2017

Abstract

Reversal learning can be used to examine deficits in cognitive flexibility, which have been linked to a number of neuropsychiatric disorders including schizophrenia and addiction. However, methods of examining reversal learning have varied substantially between species. Touchscreen technology has allowed researchers to explore cognitive deficits with a platform that is translatable across rodents, non-human primates and human subjects. Here we describe a method for measuring visual discrimination and reversal learning in mice using automated touchscreen-based operant chambers.

Keywords: Visual discrimination (视觉辨别), Reversal (颠倒), Touchscreen (触摸屏), Cognition (认知), Mice (小鼠)

Background

Cognitive flexibility is the ability to flexibly adjust responses to a previously learned stimulus-reward association, and impairments occur in a range of neuropsychiatric conditions, including schizophrenia, autism, obsessive-compulsive disorder and addiction. To further study the neural mechanisms implicated in cognitive flexibility, performance of choice and reversal tasks have been used in animal models. There are a variety of methods used to measure cognitive flexibility in rodent models, however many techniques have been difficult to compare between rodent and human studies (Brigman et al., 2010). However, using touchscreens, a similar paradigm can be used to study reversal learning across species (Bussey et al., 2012; Horner et al., 2013). Trained rodents are able to discriminate visual stimuli and then successfully reverse their choice when the contingency changes. However, cognitive performance in both rats and mice has been shown to be highly strain dependent (Graybeal et al., 2014). C57BL/6J mice are a popular strain for behavioural and genetic studies, and have been used as a standard strain against which others are compared (Izquierdo et al., 2006). Meanwhile, BALB/c mice often display poor learning and cognitive performance compared to other strains (Graybeal et al., 2014). Recently, the BALB/c strain was shown to be ‘severely impaired’ in basic training, visual discrimination and reversal learning using touchscreen chambers (Graybeal et al., 2014). Therefore, we have adapted the training protocol to promote responding in an anxious mouse strain (BALB/c), where behavioural (rather than cognitive) traits may impair performance. Our results indicated that this protocol provides comparable levels of performance in the standard C57BL/6 mouse to those previously published and significantly enhanced performance of the anxious and emotionally reactive BALB/c mouse (Turner et al., 2017).

Materials and Reagents

  1. Small containers, such as 50 ml Falcon tube lids (BD Biosciences)
  2. Paper towels for cleaning chambers
  3. Our experiment used male BALB/c or C57BL/6J mice (Animal Resource Centre, Australia) at 12 weeks of age one week after arrival to allow the mice to habituate to the facility
    Notes:
    1. Mice are housed in groups of four in individually ventilated cages (OptiMICE, Animal Care Systems, USA) with water available ad libitum. Mice are housed with bedding with tissues. The temperature (21 ± 1 °C) and humidity (50 ± 10%) are controlled and lights are kept on a 12-h cycle (lights on at 07:00 AM).
    2. Mice are tail marked for identification and weighed for 3 days to get an average free-feeding body weight. Food restriction should be conducted for at least 3 days prior to testing to gradually reduce weight and allow mice to adapt to a set feeding schedule. Food restriction is used as mice will readily perform for strawberry milk rewards when hungry.
    3. They are then food restricted to around 90% of their free-feeding body weight using small pieces of food to minimise fighting.
      Ensure that growth relevant to the strain and age is considered in determining ongoing food restriction limits.
    4. Treatment group size should be based on a power analysis where possible, however groups of 10-15 mice are commonly considered sufficient.
  4. Undiluted strawberry milk (Breaka, Parmalat, Australia)
  5. Ethanol (70%) for cleaning chambers

Equipment

  1. Bussey-Saksida Mouse Touchscreen Chambers (Campden Instruments, model: Model 80614 ) equipped with:
    1. Touchscreen
    2. House light
    3. Liquid reward dispenser
    4. Magazine
    5. Two-window black masks
    6. Overhead camera
    7. Sound-attenuating chamber
    8. Multi-Media Single Station Licence for each chamber (Campden Instruments, model: Model 80698-1 )

Software

  1. PC with ABET II Software for Touchscreens (Model 89505, Campden Instruments Ltd., UK) with Whisker Multi-Media Single Station Licence for each chamber (Model 80698-1, Campden Instruments Ltd., UK)
  2. SPSS (ver.20, SPSS Inc., Chicago, USA)

Procedure

Animal Ethics Approval: Researchers must determine ethical approvals required and maintain compliance with relevant university and government guidelines regarding the care and use of laboratory animals prior to starting experiments.

  1. Operant training
    1. Prior to training, mice are exposed to the strawberry milk reward by providing small amounts (~0.5 ml) in the home cage. A small autoclavable container (e.g., 50 ml Falcon tube lid) was half filled with milk and placed in the home cage for three consecutive days before training commenced. Mice may bury the lids rather than consume the milk initially, however three days seems sufficient to introduce the reward and reduce neophobia.
    2. Prior to transporting the mice the operant chamber equipment should be checked and prepared for testing. The mask should be placed over the touchscreens and the reward lines should be primed with fresh strawberry milk.
    3. Mice are then transported to the dimly lit (< 10 lux) testing room in their home cages and allowed 30 min to habituate each day before testing.
    4. The data and program are loaded on the computer, and then each mouse is placed into the correct operant chamber for testing. Mice are always tested in the same operant chamber because contextual changes can impair learning. Testing should occur at the same time each day and for at least 5 days per week. Feeding should also occur at the same time each day, but at least 1 h after testing has finished.

  2. Training stages
    Several steps (5 pre-training and 2 task steps) are used during training. Mice can pass to the next step if the criteria were fulfilled. The training parameters and criteria to move to the next stage are shown in Table 1. The image that is rewarded should be counterbalanced within each treatment group, but constant for an individual mouse. Within each group, half of the mice are required to respond to one or the other stimulus to receive rewards. We have used one session per day, but multiple sessions can be run in a day, depending on the number of animals in a cohort or on the number of chambers that are available. Therefore, criteria are based on the number of sessions completed.

    Table 1. Parameters and criteria for the training stages


    1. Pre-training: Habituation
      The mice learn to collect rewards from the illuminated magazine. The reward is pumped for 6,000 msec (approx. 150 μl) and the magazine light is illuminated. Once the head entry is detected, the light turns off and after the head is removed the inter-trial interval (ITI) begins (10 sec). After the ITI, a single reward (280 msec) is delivered and the magazine light illuminated, start the cycle again (Figure 1.1).
    2. Pre-training: Initial Touch
      The mouse is rewarded for touching the white square on the touchscreen. Two images are selected pseudorandomly to appear in each location. One image is a white square and the other is a blank, black screen. If the square is touched within 30 sec of display, it is removed and 3 x reward (840 msec) is delivered to an illuminated magazine. If there are no touches for 30 sec, then a single reward is delivered instead (280 msec). In both cases, removal of the head from the magazine starts the ITI (5 sec) before the next trial starts. Touches to the blank image are recorded but have no consequences (Figure 1.2).
    3. Pre-training: Must Touch
      The mice must touch the white square on the touchscreen to receive a reward. As for Must Touch, a white square will appear in one location and the mouse must touch it to receive a single reward and no reward is given for omitting a response. Blank touches have no consequences (Figure 1.3).
    4. Pre-training: Must Initiate
      The mice must start the trials by nose poking in the magazine. The protocol starts as per Must Touch with the presentation of the same images where touching the white square is rewarded and blank touches have no consequences. After reward collection, the magazine light is illuminated again and a head entry is required to proceed to the next trial (Figure 1.4).
    5. Pre-training: Punish Incorrect
      The mice receive a time-out for touching the incorrect location. As per Must Initiate, however now touching the blank image results in a time-out (5 sec) when the house light is illuminated (Figure 1.5).
    6. Task: Visual Discrimination (VD)
      Mice must discriminate between two visual stimuli and respond to the correct image. The protocol is very similar to Punish Incorrect, except that the testing stimuli pair is now used (Figure 1.6). The stimuli used have black backgrounds with either a single large white circle or four small white circles in each corner of the image (Figure 2).


      Figure 1. Visual discrimination and reversal training stages. 1. Habituation where the mouse learns to collect rewards; 2. Initial touch where the mouse is rewarded with 3 rewards for touching the white square on the screen or 1 reward after 30 sec. 3. Must touch where the mouse will only receive a reward for touching the white square on the screen. 4. Must initiate where the mouse must start trials with a magazine nose poke. 5. Punish incorrect where the mouse now receives a time-out for incorrectly touching the blank screen. 6. Visual discrimination where the mouse learns to select the correct image (large circle in example) to receive a reward. 7. Reversal where the mouse must now select the alternative image (small circles in example) to receive a reward. 8. Photograph of top view of operant chamber to show stimulus panel towards top of the image and reward collection port (indicated by white arrow).


      Figure 2. Visual discrimination stimuli. The images used in this protocol were a single large white circle versus four small white circles. These stimuli were selected as they have similar graphic features and would allow the use of orientation shifted patterns (such as vertical vs. horizontal lines) within a battery of other tasks.

    7. Task: Reversal
      The stimuli that were previously paired with reward should now be avoided and the alternative stimuli (previously unrewarded stimuli) are now correct (Figure 1.7).

Data analysis

Statistical analysis is conducted using SPSS (ver.20, SPSS Inc., Chicago, USA) with significance set at P < 0.05. The primary measures include trials completed, response accuracy (correct, incorrect or blank touch), the number of correction trials, session duration, response and reward latencies, response omission and the number of sessions required on each training stage to reach criteria. Correction trials repeat the previous stimulus in the same location until the correct stimulus/location is selected. This is very important when first training VD to prevent the mouse from simply selecting one location and being rewarded on 50% of trials. Other measures include irrelevant touches to the screen during the inter-trial interval (ITI) or time-out periods, and the number of fronts and rear beam breaks as a measure of activity. The effects of mouse strain, drug treatment or other manipulation on these measures can be examined by independent or paired t-tests, ANOVA or repeated measures ANOVA where appropriate to the experimental design and with consideration for multiple comparisons.

Notes

Representative results from two mouse strains (examples in Videos 1 and 2) can be seen in Table 2. The average number of sessions to reach criteria are given for each of the stages (5 pre-training and 2 task steps). Under these testing conditions, BALB/c mice take fewer sessions to complete the task. However, the percent correct responses for VD and reversal and the reward latency did not differ between the mouse two strains.

Video 1. Example of a C57BL/6 mice performing visual discrimination

Video 2. Example of a BALB/c mice performing reversal

Table 2. Example of results from two commonly used mouse strains

Acknowledgments

This procedure was adapted from published protocols (Izquierdo et al., 2006; Horner et al., 2013; Graybeal et al., 2014) and full results of the mouse strain comparison using this protocol can be found in Turner et al. (2017). This work was funded by the National Health and Medical Research Council of Australia and a University of Queensland Major Equipment and Infrastructure Grant to TB. KT was supported by the Queensland Government Smart Futures PhD Scholarship and an Australian Postgraduate Award. Figures were produced by Nick Valmas.

References

  1. Brigman, J. L., Graybeal, C. and Holmes, A. (2010). Predictably irrational: assaying cognitive inflexibility in mouse models of schizophrenia. Front Neurosci 4.
  2. Bussey, T. J., Holmes, A., Lyon, L., Mar, A. C., McAllister, K. A., Nithianantharajah, J., Oomen, C. A. and Saksida, L. M. (2012). New translational assays for preclinical modelling of cognition in schizophrenia: the touchscreen testing method for mice and rats. Neuropharmacology 62(3): 1191-1203.
  3. Graybeal, C., Bachu, M., Mozhui, K., Saksida, L. M., Bussey, T. J., Sagalyn, E., Williams, R. W. and Holmes, A. (2014). Strains and stressors: an analysis of touchscreen learning in genetically diverse mouse strains. PLoS One 9(2): e87745.
  4. Horner, A. E., Heath, C. J., Hvoslef-Eide, M., Kent, B. A., Kim, C. H., Nilsson, S. R., Alsio, J., Oomen, C. A., Holmes, A., Saksida, L. M. and Bussey, T. J. (2013). The touchscreen operant platform for testing learning and memory in rats and mice. Nat Protoc 8(10): 1961-1984.
  5. Izquierdo, A., Wiedholz, L. M., Millstein, R. A., Yang, R. J., Bussey, T. J., Saksida, L. M. and Holmes, A. (2006). Genetic and dopaminergic modulation of reversal learning in a touchscreen-based operant procedure for mice. Behav Brain Res 171(2): 181-188.
  6. Turner, K. M., Simpson, C. G. and Burne, T. H. (2017). BALB/c mice can learn touchscreen visual discrimination and reversal tasks faster than C57BL/6 mice. Front Behav Neurosci 11: 16.

简介


材料和试剂




  1. Microtube(INA•OPTIKA,Bio-Bik,目录号:ST-0150F)



  2. 多孔培养板6孔(Greiner Bio One International,目录号:657160)



  3. 15ml锥形离心管(Greiner Bio One International,目录号:188271)



  4. 具有0.22μm孔径膜的注射器过滤器(Pall,目录号:4192)



  5. HEK293T细胞



  6. Notch1表达载体(pTracer-CMV / Notch1 )(Sawaguchi等人,2017)

    注意:不可商业使用。



  7. 表达载体(pSecTag2 / Hygro / Eogt)的Eogt表达载体(Sawaguchi等人,2017)

    注意:不可商业使用。



  8. GFP表达载体(pMAX-GFP)(Addgene,目录号:VDF-1012)



  9. Dynabeads蛋白A(Thermo Fisher Scientific,Invitrogen TM,目录号:10002D)



  10. Opti-MEM(Thermo Fisher Scientific,Gibco TM ,目录号:31985070)



  11. 4%多聚甲醛(和光纯药,目录号:163-20145)



  12. 胎牛血清(FBS)(Sigma-Aldrich,目录号:172012-500ML)



  13. 磷酸二氢钠(Na 2 HPO 4)(和光纯药,目录号:196-02835)



  14. 磷酸二氢钾(KH 2 PO 4)(和光纯药,目录号:164-04295)



  15. 氯化钠(NaCl)(Wako Pure Chemical Industries,目录号:197-01667)



  16. 氯化钾(KCl)(Wako Pure Chemical Industries,目录号:163-03545)



  17. DLL4-Fc(Thermo Fisher Scientific,目录号:10171H02H)



  18. JAG1-Fc(Thermo Fisher Scientific,目录号:11648H02H)



  19. Dulbecco改良Eagle培养基(NISSUI PHARMACEUTICAL,目录号:05915)



  20. 青霉素 - 链霉素(Thermo Fisher Scientific,Gibco TM,目录号:15140122)



  21. 聚乙烯亚胺,线性,MW 25,000(PEI 25000)(Polysciences,目录号:23966)



  22. 胎牛血清(FBS)(见食谱)



  23. 磷酸缓冲盐水(PBS)(见食谱)



  24. 100ng /μlDLL4-Fc / PBS和100ng /μlJAG1-Fc / PBS(参见食谱)



  25. 完成文化媒体(见食谱)



  26. PEI解决方案(见配方)




设备




  1. 移液器(各种尺寸)(GILSON)



  2. 6管磁选机架(New England Biolabs,目录号:S1506S)



  3. 管旋转器(Taiyo,型号:RT-50)(图1A)



  4. CO 2培养箱(SANYO,目录号:MCO-175)



  5. 轨道旋转器(东方仪器,目录号:KS-6300)(图1B)



  6. 自制吸气器(图1C和1D)



  7. 盒式荧光成像装置(Olympus,目录号:FSX100)





    图1.设备。 A.管旋转器RT50;轨道旋转器C和D.自制吸气器。




程序




  1. 配体涂层Dynabeads的制备


    1. 将20μlDynabeads蛋白A用1ml PBS(见食谱)在微管中洗涤。

      注意:标准程序可以针对多个样本进行放大。



    2. 使用磁分离架收集珠粒。



    3. 将溶液从珠粒中取出。



    4. 将珠子重新悬浮于100μlPBS中


    5. 将2μl100ng /μl的DLL4-Fc / PBS(参见食谱)或JAG1-Fc / PBS(参见食谱)加入到管中



    6. 珠子在室温下孵育20分钟或在4℃下过夜,使用管旋转器(视频1)。




    7. 使用磁选机架收集珠粒。



    8. 将溶液从珠粒中取出。



    9. 用1ml PBS洗涤珠子3次。



    10. 将溶液从珠粒中取出。



    11. 将100μlPBS加入管中。



    12. 在使用前,将900μl冰冷的完整培养基加入管中






  2. Notch表达细胞的制备


    1. 将HEK293T细胞以6×10 5个细胞/孔接种在6孔板中,在完全培养基中(参见食谱)。



    2. 第二天,用800μl的Opti-MEM代替培养基。



    3. 将细胞在37℃下在CO 2培养箱中孵育30分钟


    4. 将质粒DNA(2μg)在微管中稀释成200μl的Opti-MEM
      注意:为了鉴定转染的细胞,将GFP表达载体(例如,pMAX-GFP)包含在1/8总量质粒中。



    5. 管子轻轻旋涡。



    6. 将6μl1mg / ml PEI溶液(参见食谱)加入到DNA溶液中


    7. 混合物在室温下孵育30分钟


    8. 将DNA / PEI混合物轻轻滴在板的每个孔上


    9. 将细胞在37℃下在CO 2培养箱中孵育4小时


    10. 用2ml的完整培养基代替培养基。



    11. 将细胞在37℃下在CO 2培养箱中孵育48小时






  3. 结合测定(图2)





    图2.结合测定。 A.在15ml管中完成培养基;将动物重悬于完全培养基中;将6孔板中的HEK293T细胞与冷室中的配体偶联珠孵育。 D.用多聚甲醛固定培养板; E.去除PBS前的培养板;使用自制抽吸器从培养板中取出PBS。 G.去除PBS后的培养板; H.用PBS洗涤培养板三次; I.用PBS洗涤五次后的培养板。





    1. 用含有DLL4-Fc或JAG1-Fc珠的1ml冰冷的完全培养基代替培养基(图2C)。



    2. 细胞在冷室中孵育30分钟


    3. 使用吸气器将培养基从培养皿中取出


    4. 细胞在室温下用4%多聚甲醛固定20分钟(图2D)


    5. 使用轨道旋转器将固定的细胞用5ml PBS洗涤5分钟(视频2)



    6. 使用吸气器将PBS从培养皿中取出(视频3)
      注意:可以使用吸气器将积聚在每个孔中心的珠子移除。




    7. 通过重复步骤C5和C6,将细胞洗涤5次。



    8. 将1ml PBS加入到培养皿的每个孔中(图2I)


    9. 使用FSX100捕获相位对比度和荧光图像(图3)


    10. 计数GFP阳性细胞上结合的珠粒数(图4)
      注意:计数50个GFP阳性细胞进行定量。排除浮珠以进行计数。





      图3. Dynabeads绑定单元格的计数。 A-C。转染表达GFP的HEK293T细胞用对照Dynabeads培养。 D-F。将转染表达GFP,Notch1的HEK293T细胞与DLL4-Fc结合的珠孵育。 G-我。将转染以表达GFP,Eogt和Notch1的HEK293T细胞与DLL4-Fc结合的珠孵育。 A,D和G.相位图像; B,E和H.荧光图像; C,F和I.合并图像。刻度棒=60μm。 C',F'和I'。图像中盒装区域的放大倍数(C,F,I)。刻度棒=60μm。 C“,F”和I“。与C',F'和I'相同。红点表示GFP阳性细胞上的结合珠。虚线显示细胞的轮廓。刻度棒=60μm。





      图4. Dynabeads绑定细胞的计数分析。 HEK293T细胞或Notch1瞬时转染有或没有Eogt 的细胞与DLL4或JAG1珠孵育。确定用GFP表达标记的转染细胞结合的Dynabeads数(n = 50)。数据是来自三个独立实验的平均值±SD。 * P &lt; 0.05; ** 0.01; Welch的测试条。






数据分析



在先前发表的实验(Sawaguchi等人,2017)中,数据显示为来自三个独立实验的平均值±SD。在每个实验中,分析了50个GFP阳性细胞。 Welch的 t -test被使用。



笔记



该协议提供高重现性。计数50个GFP阳性细胞在大多数实验中提供了低变异性。



食谱




  1. 胎牛血清(FBS)

    将含有FBS的瓶子在56℃下孵育30分钟,然后使用



  2. 磷酸盐缓冲盐水(PBS)

    10mM Na 2 HPO 4

    1.8mM KH PO 4

    137 mM NaCl

    2.7 mM KCl



  3. 100ng /μlDLL4-Fc / PBS和100ng /μlJAG1-Fc / PBS
    将DLL4-Fc或JAG1-Fc(5μg)溶于50μlPBS

    注意:100ng /μlDLL4-Fc / PBS和100ng /μlJAG1-Fc / PBS可以在4℃下储存3个月。



  4. 完成文化媒体

    含有10%FBS和青霉素 - 链霉素的DMEM


  5. PEI解决方案


    1. 将100毫升PEI溶于100ml Milli-Q水中


    2. 溶液通过0.22μm的膜进行灭菌


    3. 等分试样储存于-30°C






致谢



该协议已经从之前发表的文章(Sawaguchi等人,2017年)进行了修改。这项工作得到日本社会科学促进会授予#JP15K15064 TO和MO,#JP26110709 to TO,#JP26291020 to TO,#JP15K18502 to MO,#JP16J00004 to MO;武田科学基金会日本应用酶学基金会TO TO; YOKOYAMA临床药理学基金会#YRY-1612至MO。



参考




  1. Moloney,DJ,Panin,VM,Johnston,SH,Chen,J.,Shao,L.,Wilson,R.,Wang,Y.,Stanley,P.,Irvine,KD,Haltiwanger,RS和Vogt,TF(2000 )。 边缘是修改Notch的糖基转移酶。 自然 406 (6794):369-375。



  2. Mumm,J.S。和Kopan,R。(2000)。 Notch信号:来自外部。 Dev Biol 228(2):151-165。



  3. Sawaguchi,S.,Varshney,S.,Ogawa,M.,Sakaidani,Y.,Yagi,H.,Takeshita,K.,Murohara,T.,Kato,K.,Sundaram,S.,Stanley,冈岛,T.(2017)。 NOTCH1上的O-GlcNAc EGF重复调节哺乳动物中配体诱导的Notch信号传导和血管发育。 a> Elife 6.



  4. Stanley,P.和Okajima,T。(2010)。
    Notch信号传导中糖基化的作用。 Curr Top Dev Biol 92 : 131-164.




关键字:视觉辨别, 颠倒, 触摸屏, 认知, 小鼠

材料和试剂

  1. 小容器,如50ml Falcon管盖(BD Biosciences)
  2. 用于清洁室的纸巾
  3. 我们的实验在到达后一周在12周龄时使用雄性BALB / c或C57BL / 6J小鼠(澳大利亚动物资源中心),以使小鼠习惯于设施
    注意:
    1. 小鼠在四个独立通风的笼子(OptiMICE,动物护理系统,美国)中放置,随意供水。小鼠被安置在床上用纸巾。温度(21±1℃)和湿度(50±10%)被控制,灯保持12小时循环(07:00 AM点亮)。
    2. 小鼠尾部标记识别,称重3天,获得平均自由体重。食品限制应在测试前至少3天进行,以逐渐减轻体重,并允许小鼠适应一定的进食时间表。使用食物限制,因为当饥饿时,小鼠将很容易为草莓奶奖励。















    3. 确保在确定持续的食品限制限度时考虑与应变和年龄相关的增长。
    4. 治疗组大小应尽可能基于功率分析,但10-15只小鼠的群体通常被认为是足够的。
  4. 未稀释的草莓牛奶(Breaka,Parmalat,澳大利亚)
  5. 用于清洁室的乙醇(70%)

设备

  1. Bussey-Saksida鼠标触摸屏室(Campden Instruments,型号:80614型)配备:
    1. 触摸屏
    2. 屋灯
    3. 液体报酬分配器
    4. 杂志
    5. 双窗口黑色口罩
    6. 架空摄像头
    7. 声音衰减室
    8. 每个房间的多媒体单站许可证(Campden Instruments,型号:80698-1)

软件

  1. 具有用于触摸屏的ABET II软件(型号89505,Campden Instruments Ltd.,英国)的每个室的Whisker多媒体单站许可证(型号80698-1,Campden Instruments Ltd.,UK)
  2. SPSS(ver.20,SPSS Inc.,Chicago,USA)

程序

动物伦理批准 :研究人员必须确定所需的伦理认证,

  1. 操作培训
    1. 在训练之前,通过在家庭笼中提供少量(约0.5ml),将小鼠暴露于草莓奶的报酬。将一小部分可高压灭菌的容器(例如,50ml Falcon管盖)一半填充牛奶,并在培训开始前放置在家庭笼中连续三天。小鼠最初可能会掩盖盖子而不是消费牛奶,但是三天似乎足以引进奖励并减少恐惧症。
    2. 运输小鼠之前,应检查手术室设备以进行测试。面罩应放在触摸屏上,奖励线应该用新鲜的草莓牛奶灌注。
    3. 然后将小鼠运送到家庭网箱中的昏暗(<10 lux)检测室,并允许在测试前每天休息30分钟。
    4. 将数据和程序加载到计算机上,然后将每只鼠标放入正确的操作室进行测试。小鼠总是在相同的操作室中测试,因为上下文的变化可能会损害学习。测试应每天同时进行,每周至少5天。喂食也应每天同时进行,但至少1小时后测试完成。

  2. 培训阶段
    在训练过程中使用了几个步骤(5个预训练和2个任务步骤)。如果满足标准,小鼠可以转到下一步。移动到下一阶段的训练参数和标准如表1所示。奖励的图像应在每个治疗组内平衡,但对于单个鼠标而言是恒定的。在每组中,一半的小鼠需要响应一种或另一种刺激来获得奖励。我们每天使用一个会话,但是可以在一天内运行多个会话,具体取决于队列中的动物数量或可用的数量。因此,标准是基于完成的会话数量。

    表1.培训阶段的参数和标准


    1. 预训练:习惯性
      老鼠学会从照明杂志收集奖励。奖励被抽取6,000毫秒(约150微升),并且杂志灯被照亮。一旦检测到头部入口,灯熄灭,并且在头部被移除之后,试验间隔(ITI)开始(10秒)。在ITI之后,交付单次奖励(280毫秒),并且杂志灯亮起,再次开始循环(图1.1)。
    2. 预训练:初步触摸
      鼠标在触摸屏上触摸白色方块的奖励。两个图像被伪随机选择出现在每个位置。一个图像是一个白色方块,另一个是空白的黑色屏幕。如果在显示的30秒内触摸正方形,则将其移除,并将3 x奖励(840毫秒)传送到照明杂志。如果没有30秒的触摸,则会传送单个奖励(280毫秒)。在这两种情况下,在下次试用开始之前,从刀库中取出刀头将启动ITI(5秒)。记录空白图像的记录,但没有后果(图1.2)。
    3. 预训练:必须触摸
      小鼠必须触摸触摸屏上的白色方块才能获得奖励。至于必须触摸,一个白色方块将出现在一个位置,鼠标必须触摸它才能获得单个奖励,并且不给予奖励以省略响应。空白的事情没有后果(图1.3)。
    4. 预训练:必须启动
      小鼠必须通过鼻子戳在杂志上开始试验。协议按照必须触摸开始,同时呈现相同的图像,触摸白色方块即可获得奖励,空白触摸也没有后果。收集奖励后,杂志光再次亮起,头条目需要进入下一次试验(图1.4)。
    5. 预训练:惩罚不正确
      鼠标接收到不正确位置的超时。按照必须启动,然而触摸空白图像会导致室内灯亮起的超时(5秒)(图1.5)。
    6. 任务:视觉歧视(VD)
      小鼠必须区分两种视觉刺激并对正确的图像做出反应。该协议非常类似于惩罚不正确,除了现在使用测试刺激对(图1.6)。使用的刺激具有黑色背景,其中每个角落中都有一个大的白色圆圈或四个小的白色圆圈(图2)。


      图1.视觉歧视和反转训练阶段。 1。老鼠学习收集奖励的习惯; 2.初始触摸,鼠标在屏幕上触摸白色方块3次奖励,30秒后获得1次奖励。 3.必须触摸鼠标只能在触摸屏幕上的白色方块上获得奖励。 4.必须启动鼠标必须开始使用杂志鼻捅的试验。 5.处理不正确的地方,鼠标现在收到超时以正确触摸空白屏幕。 6.视觉区别,鼠标学习选择正确的图像(例如大圆圈)以获得奖励。 7.反转,鼠标现在必须选择替代图像(例如小圆圈)来获得奖励。 8.操作室顶视图,向图像顶部显示刺激面板和奖励收集口(白箭头所示)。


      图2.视觉辨别刺激。 此协议中使用的图像是一个大的白色圆圈,而不是四个小的白色圆圈。选择这些刺激物,因为它们具有相似的图形特征,并允许在一系列其他任务中使用取向移动的图案(例如垂直对水平线)。

    7. 任务:冲销
      以前配合奖励的刺激现在应该被避免,替代刺激(以前没有被刺激)现在是正确的(图1.7)。

数据分析

统计分析使用SPSS(ver.20,SPSS Inc.,Chicago,USA)进行,其显着性设置为P < 0.05。主要措施包括审核完成,回复准确性(正确,不正确或空白),纠正试验次数,会话持续时间,回复和奖励延迟,回应遗漏以及每个培训阶段达到标准所需的会话次数。校正试验在同一位置重复先前的刺激,直到选择了正确的刺激/位置。这是非常重要的,当第一次训练VD,以防止鼠标选择一个位置,并获得50%的审判奖励。其他措施包括在试用间隔期间(ITI)或超时期间对屏幕的无关触摸,以及作为活动量度的前端和后横梁断裂数。小鼠应变,药物治疗或其他操作对这些措施的影响可以通过独立或配对的试验,方差分析或重复测量方差分析来检查,适用于实验设计并考虑多重比较。

笔记

表2中可以看到两个小鼠品系的代表性结果(视频1和2中的示例)。为每个阶段(5个预训练和2个任务步骤)给出达到标准的平均会话次数。在这些测试条件下,BALB / c小鼠需要较少的会话才能完成任务。然而,对于VD和逆转的百分比正确反应和小鼠两株之间的回报延迟没有不同。

表2.来自两个常用的小鼠株的结果实例

致谢

该程序根据已公布的方案(Izquierdo等人,2006; Horner等人,2013; Graybeal等人,2014)进行了改编,并且使用该方案的小鼠应变比较的全部结果可以在Turner等人(2017)中找到。这项工作由澳大利亚国家卫生和医学研究理事会和昆士兰大学主要设备和基础设施获得资助。 KT获得昆士兰政府智慧未来博士奖学金和澳大利亚研究生奖。数字由尼克•瓦尔马斯(Nick Valmas

参考

  1. Brigman,J.L.,Graybeal,C.and Holmes,A。(2010)。 预测性非理性:测定精神分裂症小鼠模型中的认知不灵活性。前Neurosci 4。
  2. Bussey,T.J.,Holmes,A.,Lyon,L.,Mar,A.C.,McAllister,K.A.,Nithianantharajah,J.,Oomen,C.A。和Saksida,L.M。(2012)。 精神分裂症认知临床前建模的新翻译测定:小鼠和大鼠的触屏测试方法。 / a>神经药理学 62(3):1191-1203。
  3. Graybeal,C.,Bachu,M.,Mozhui,K.,Saksida,L.M.,Bussey,T.J.,Sagalyn,E.,Williams,R.W.and Holmes,A。(2014)。 菌株和压力源:分析基因多样性小鼠品系中的触屏学习。 > PLoS One 9(2):e87745。
  4. Horner,AE,Heath,CJ,Hvoslef-Eide,M.,Kent,BA,Kim,CH,Nilsson,SR,Alsio,J.,Oomen,CA,Holmes,A.,Saksida,LM and Bussey,TJ )。 用于测试老鼠和小鼠学习和记忆的触摸屏操作平台 Nat Protoc 8(10):1961-1984。
  5. Izquierdo,A.,Wiedholz,L.M.,Millstein,R.A.,Yang,R.J。,Bussey,T.J.,Saksida,L.M。和Holmes,A。(2006)。 基于触摸屏的小鼠手术过程中逆转学习的遗传和多巴胺能调节。 Behav Brain Res 171(2):181-188。
  6. Turner,K.M.,Simpson,C.G。和Burne,T.H。(2017)。 BALB / c小鼠可以比C57BL / 6小鼠更快地学习触摸屏视觉辨别和逆转任务。 a> Front Behav Neurosci 11:16.
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Turner, K. M., Simpson, C. and Burne, T. (2017). Touchscreen-based Visual Discrimination and Reversal Tasks for Mice to Test Cognitive Flexibility. Bio-protocol 7(20): e2583. DOI: 10.21769/BioProtoc.2583.
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