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Olfactory Cued Learning Paradigm
嗅觉暗示学习范例   

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Abstract

Sensory stimulation leads to structural changes within the CNS (Central Nervous System), thus providing the fundamental mechanism for learning and memory. The olfactory circuit offers a unique model for studying experience-dependent plasticity, partly due to a continuous supply of integrating adult born neurons. Our lab has recently implemented an olfactory cued learning paradigm in which specific odor pairs are coupled to either a reward or punishment to study downstream circuit changes. The following protocol outlines the basic set up for our learning paradigm. Here, we describe the equipment setup, programming of software, and method of behavioral training.

Keywords: Olfactory(嗅觉), Circuit(电路), Learning(学习), Synaptic(突触), Plasticity(可塑性), Go/No-Go(Go/No-Go), Behavior(行为)

Background

The adult brain features ongoing experience-dependent structural changes. Within the rodent olfactory bulb (OB) where odor information is first processed, a continuous supply of adult born interneurons (granule cells) either integrates into the olfactory circuitry or undergoes apoptosis (Petreanu and Alvarez-Buylla, 2002; Carleton et al., 2003; Lledo et al., 2006; Sakamoto et al., 2014). This choice between survival or death is greatly influenced by sensory stimulus and olfactory cued learning (Rochefort et al., 2002; Alonso et al., 2006). Moreover, younger granule cells also undergo experience-dependent synaptic changes within a critical time window (Yamaguchi and Mori, 2005). To examine how sensory experience affects synaptic plasticity in OB circuits, our lab has successfully implemented a Go/No-Go olfactory cued learning task (Huang et al., 2016; Quast et al., 2016). Mice are trained to associate a ‘Go Odor’ with a water reward and a separate ‘No-Go Odor’ with a punishment (trial timeout) (Figure 1). Upon completion of training, mice will be able to distinguish the two odors by performing the associated task with greater than 85% accuracy (Supplemental Video 1).


Figure 1. Go/No-Go task. Trained, water-deprived mice will first poke their nose into the central odor port to initiate odor delivery. Subsequently, either a Go or No-Go odor is delivered at random. If the Go Odor is delivered, trained mice will move to either of the two side ports to collect the water reward. If the No-Go odor is delivered, trained mice will refrain from seeking water and re-poke into the odor port.

Materials and Reagents

  1. Distinct pair of odorants selected by experimenter to represent the ‘Go’ or S+ stimulus and ‘No-Go’ or S- stimulus. Example: 1-butanol and propionic acid (Sigma-Aldrich, catalog numbers: 437603 and 402907 , respectively)–diluted to 10% in mineral oil (Alfa Aesar, catalog number: 31911 ) (500 µl odorant in 5 ml of mineral oil)
  2. Qorpak borosilicate glass vial with Green Polypropylene Hole Cap (Qorpak, catalog number: GLC-01016 )
  3. Nalgene silicone tubing (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 8600-0030 )
  4. BD PrecisionGlideTM 18 gauge beveled needles (BD, catalog number: 305196 )
  5. Adult mice (> 6 weeks old, our lab uses on average 12 to 19 weeks old mice with average body weights of 19 g for females and 23 g for males)

Equipment

  1. Extra Wide Modular Test Chamber-Mouse (Med Associates, catalog number: ENV-307W )
  2. Stainless Steel Grid Floor (Med Associates, catalog number: ENV-307W-GFW )
  3. Illuminated Nose Poke Response for Wide Mouse Modular Chamber (Med Associates, catalog number: ENV-313W ) (x2)
  4. Two Channel Olfactory Stimulus (Med Associates, catalog number: ENV-275 )
  5. Illuminated Nose Poke Response with Olfactory Ports for Mouse (Med Associates, catalog number: ENV-375W-NPP )
  6. Stand Alone USB Interface, 4 In/8 out Compatible with 32bit OS only (Med Associates, catalog number: DIG-703A-USB )
  7. Standard desktop computer with Windows 2000, XP, Vista, or 7 operating system
  8. VWR flow meter, Acrylic (VWR, catalog number: 97004-952 )


    Figure 2. Go/No-Go equipment. The behavior system contains 6 key components: Behavior chamber, water reservoirs, air adjustment, USB interface system, odor delivery module, and a personalized computer (A). Room air is first relayed from the air adjustment to the input air manifold and subsequently diverged to the two odor-containing glass vials and the center valve (B). Odorized air outputs are controlled by two solenoids (C), which can be programmed by the Schedule Manager software. An odor intake line, a vacuum line, and two water dispensers are connected to the behavior chamber (D). Each odor vial is paired with its own two silicone tubes fitted with 18 gauge needles to prevent contamination of odors (E).

Software

  1. MED-PC IV behavioral software suite (Med Associates, SOF-735)
    Note: This version of the software has now been replaced with Med-PC V.
  2. MPC2XL-Data Transfer Utility for all MED-PC Users (Med Associates, SOF-731), required for data reformatting to Excel
  3. Schedule Manager Software (Med Associates, catalog number: DIG-703A-USB), required for programming training stages

Procedure

  1. Establishing behavioral chamber and programmed olfactory cue and reward delivery
    1. Obtain and set up operant conditioning behavioral chamber for mice
      1. Set up must include modular odorant and water delivery ports.
      2. Programming training stages for associative learning requires a computer that can run Med-PC IV software.
      3. Med-PC IV software utilizes a proprietary programming language that the experimenter must learn to program training stages. Specific requirements for each stage of training include:
        1. For all stages: Trials are blocked into 20 trials per block to compute per-block performance of each mouse.
        2. Stage 1: The program must specify that the mouse receives a water reward when its nose pokes into the center water delivery port. Output parameters for this stage are: time duration of trial and number of rewards received.
        3. Stage 2: The program must specify that the mouse receives water reward from two side ports only after it pokes its nose into the odor delivery port (center port). Water should be dispensed immediately after center poke even if the mouse does not go to the side ports for the reward. Output parameters: time duration of trial, number of trials initiated and number of timely rewards (received within 5 sec of a nose poke in the center port).
        4. Stage 3: The program must specify the delivery of the Go (S+) odor upon nose poke to the center port. The water reward should be only dispensed if mouse nose pokes the side ports within 5 sec of odor delivery. The program must also automatically shape the behavior of mice with a 50 msec step-wise increase (from 100-400 msec) in the length of time required for a center port nose poke to yield a reward. If within a block of 20 trials the mouse pokes in the center port long enough 80% of the time, the program should increase the required time by 50 msec. Output parameters are identical to those in stage 2 plus number of rewards received and percent correct response in all trials.
        5. Stage 3: Pseudo-training: This program is utilized for control groups of mice that undergo training without association of Go or No-Go (S-) odors to either reward or punishment. All parameters are the same as normal stage 3 except S+ odor will not be introduced.
        6. Stage 4A: At this stage, the program must start with all trials delivering Go/S+ odorant like stage 3. When mice complete 1-2 blocks of Go/S+ trials with > 85% accuracy, the experimenter can program randomized delivery of the S-/Go odor for half of the trials. 2 sec ‘time out’ punishment will be given if the mouse inaccurately seeks a water reward after the No-Go cue. To help mice distinguish between Go and No-Go trials, the experimenter can program the side lights on the chamber near the ports to be on during Go trials and off during No-Go trials. Output parameters are: Time duration of trial, Number of trials initiated (Number of center port nose pokes), Number of trials completed (Number of center port nose pokes longer than 400 msec), % correct response, Number of rewards received.
        7. Stage 4B: The program must increase the ‘time out’ punishment for mice to 4 sec. Output parameters are identical to those in stage 4A.
        8. Stage 4: Pseudo-training: Same as stage 4B except reward and punishment are assigned randomly to Go/S+ and No-Go/S- odors.
        9. Go/No-Go: This stage represents ‘testing’ phase and can be performed on a novel pair of odorants to test how accurately odor associations are made for experimental versus control mice. Output parameters are identical to those in stage 4A. If side lights were programmed in stage 4A, the program should now eliminate these side light cues.

  2. Associative learning (Go/No-Go) task
    1. Preparation of mice [3 days]
      1. 3 days prior to preliminary training, water is restricted to 40 ml/kg/day and mice are habituated to the testing environment and chamber.
        1. Preliminary trials of water restriction on control animals may be performed to determine the minimum volume of water provided to maintain performance in the behavioral task without surpassing humane endpoints (National Research Council 2003) as determined by the experimenter’s IACUC guidelines. In our hands, 40 ml/kg/day per mouse sufficed to maintain performance while avoiding bodyweight loss in excess of 15% of each animal’s baseline. This endpoint was established due to the ability of rodents to compensate to a gradual water restriction paradigm, and sustain losses up to 15% of their baseline body weight, without displaying indicators of distress (Rowland, 2007)
        2. During water restriction, mice must be weighed daily to ensure that they are above 85% of baseline mass. If mice fall below this threshold, they are removed from the study and provided free water.
        3. Note that as with other behavioral paradigms, results can vary exquisitely with small changes in procedure. Therefore, before the start of training, ensure that all environmental factors (temperature, noise, stray odors) remain constant throughout the protocol and that the experimental observers do not introduce random variation (e.g., the scent of perfumes or irregular changes in observers/handling procedure).
    2. Preliminary training [8-24 days]
      1. Mice are divided into control, pseudo-trained, or trained groups.
        1. Control mice receive no further treatment.
        2. Pseudo-trained mice are treated similarly to the trained group, except each trial yields reward and punishment at equal probability regardless of the odor presented.
        3. Trained mice are subjected to a Go/No-Go olfactory associative learning paradigm.
      2. Configure the behavioral chamber to have only one central port available to deliver water. (Figure 3A)


        Figure 3. Port configurations in behavioral chamber. Stage 1 contains a center water port (A) with the side ports inaccessible. All other stages including the Go/No-Go testing stage are performed with a central odor port and two water side ports (B).

      3. Pseudo-trained and trained mice are subjected to the following phases of preliminary training.
        1. Stage 1 [1-3 days, 60 min/mouse/day]: mouse learns to associate the chamber with the presence of water.
          1) Allow each mouse to explore the chamber and discover that the port delivers water.
          2) Completion criteria: 100 trials within 60 min.
          Notes:
          1) The sound of the water valve may startle mice, and they might stop performing. If mice are too afraid to explore, ‘free’ water can be offered. This is done by using the timed output option on the software to deliver ~200 msec worth of water into the water delivery port.
          2) If a mouse freezes or stops exploring despite free water delivery as above, allow the mouse to stay in the chamber until the end of 60 min. Remove and repeat this stage on the following day for that mouse.
          3) Empty any solid waste from the mice between trials. Spray and wipe the chamber with 70% ethanol between cages of mice to prevent odor distraction.
          4) Even within groups, mice display natural variability in performance. Thus, mice can be promoted to stage 2 as a group when the average performance is within a reasonable range of the standard above (100 completed trials within 60 min).
        2. Stage 2 [1-5 days, 60 min/mouse/day]: Mice are taught to nose poke into the center port for an automatic water reward that will be dispensed from both side ports.
          1) Configure the chamber to have one central port for odor delivery and two side ports for water delivery (Figure 3B). This will be the chamber set up for the remainder of the experiment.
          2) Completion criteria: 40-50 + trials in 60 min, with at least 25-50% timely rewards. Timely rewards are those received within 5 sec of a nose poke in the center port.
        3. Stage 3 [1-3 days, 60 min/mouse/day]: Mice must first poke into the center port then the side port within 5 sec to get a reward. Further, mice must nose-poke in the center for longer and longer times (up to 400 msec) before seeking water at either of the side ports for a dispensed reward. This stage also introduces the Go/S+ odor paired with a water reward.
          1) Completion criteria: > 60 rewards within 60 min.
          2) Apply stage 3 pseudo-training to the pseudo-trained control group, but do not introduce the S+/Go odor.
        4. Stage 4A [1-2 days, 60 min/mouse/day]: This stage trains mice on the Go vs. No-Go task and introduces the No-Go/S- odor, along with a 2 sec time out punishment for the mouse seeking a water reward on the No-Go cue. The program will start with all Go/S+ odor presentation. Parameters can be switched to 50% Go/S+ and 50% No-Go/S- trial after the mouse performs well on 1-2 blocks of all Go/S+ trials.
          1) Completion criteria: 40 trials with > 60% correct responses.
          2) Apply stage 4 pseudo-training to the pseudo-trained control group.
        5. Stage 4B [5-11 days, 60 min/mouse/day]: this stage increases punishment time to 4 sec for mice incorrectly seeking rewards on a No-Go/S- trial or failing to seek a water reward on a Go/S+ trial.
          1) Completion criteria: > 100 trials within 60 min at > 85% accuracy.
          Notes:
          1) It is helpful to keep daily track of not only the total % correct, but also the max % and min % correct for each session, to monitor each mouse’s progress during this stage.
          2) In our hands, 85-90% of control mice reach the completion criteria. If the study requires that the mice continue into the Go/No Go testing phase, we recommend exclusion of mice who do not meet these criteria moving forward, as they have not demonstrated adequate ability to perform the task.
          3) The experimenter may wish to program a ‘reversal’ training trial at the end of this stage, wherein the Go and No-Go odors are swapped in their position on the odor vial platform. This is to ensure that the mice are not cueing to another stimulus (e.g., the sound of a specific valve associated with one or the other side port).
    3. Go/No-Go trial [1 day, ~20 min/mouse/day]
      1. Mice promoted beyond stage 4B of training have now sufficiently learned the behavioral task and can be exposed to novel odor pairs to test their ability to discriminate between new odors (e.g., stereoisomers such as + and - limonene).
      2. The parameters of Go/No-Go trials are like those of stage 4B, with the exception that a 300 msec nose poke in the center port is sufficient for mice to execute either the Go or No-Go action.
      3. Enter experimental details into the program. Load each mouse into the chamber and select the correct program to run from the on-screen interface.
      4. The endpoints or variables of statistical analysis are determined by the experimenter, but in our hands, control mice will often display accurate performance with new odor pairs (> 85% correct responses) after approximately 200-400 trials or 10-20 blocks.

Data analysis

In the trained group, only mice which perform the Go/No-Go trial at > 85% accuracy was utilized in our circuit mapping experiments (Huang et al., 2016). Mice that did not meet the criteria are not included in further studies. Furthermore, olfactory learning progression can be displayed for both the initial two associated odors used in training (Figure 4A), as well as for any subsequent re-associated odors (Figure 4B). Any statistical package may be used to analyze data, with specific tests depending on the experimenter’s needs. In Huang et al. (2016), connectivity patterns in the olfactory bulb among trained, pseudotrained and control groups were compared by two-way ANOVA with repeated measures.


Figure 4. Learning curve. In stage 4B, mice are presented both Go and No-Go trials at random. Average percent correct choice for sequential trial blocks (20 trials/block) are shown for a group of 5 mice (A). Error bars display standard error for the same block across all animals within one group. Re-association of novel odorants once mice are successfully trained can also be graphed similarly (B).

Notes

  1. To reduce variability between experiments, we recommend distributing a controlled mix of age and sex of experimental animals among all groups tested.
  2. Be certain that all tubing and fittings on the behavioral chamber are connected tightly to their bases to prevent odorant leakage.
  3. Set up the behavior box in a low traffic, dimly lit area to prevent distractions to the mice.
  4. Keep house air flow at ~3-5 L/min and adjust vacuum suction in the center port to avoid lingering odors between trials.
  5. Use odor-specific tubes to connect odorant vials to the behavioral chamber. This will eliminate cross-contamination of odorants.
  6. Clean the box thoroughly with 70% EtOH between cages/sets of mice.

Recipes

  1. Odor preparation
    1. Add 500 μl of odor with 4.5 ml of mineral oil into borosilicate glass vial
    2. Prepare two 1 foot length silicon tubings and fit one end of each into separate 18 gauge needles. Both needles will be placed into the odor vial (Supplemental Video 2) and the tube ends will be fitted either to the air intake or the air outlet to the odor box
    3. Always prepare new odors on the same day. Odors are remade weekly 

Acknowledgments

This protocol is adapted from previous work within our lab (Huang et al., 2016). It is supported by the McNair Medical Institute, NINDS grant R01NS078294 to B.R.A., and NIH IDDRC grant U54HD083092. The authors declare no conflicts of interest and no competing interests.

References

  1. Alonso, M., Viollet, C., Gabellec, M. M., Meas-Yedid, V., Olivo-Marin, J. C. and Lledo, P. M. (2006). Olfactory discrimination learning increases the survival of adult-born neurons in the olfactory bulb. J Neurosci 26(41): 10508-10513.
  2. Carleton, A., Petreanu, L. T., Lansford, R., Alvarez-Buylla, A. and Lledo, P. M. (2003). Becoming a new neuron in the adult olfactory bulb. Nat Neurosci 6(5): 507-518.
  3. Huang, L., Ung, K., Garcia, I., Quast, K. B., Cordiner, K., Saggau, P. and Arenkiel, B. R. (2016). Task learning promotes plasticity of interneuron connectivity maps in the olfactory bulb. J Neurosci 36(34): 8856-8871.

简介

感觉刺激导致CNS(中枢神经系统)内的结构变化,从而提供学习和记忆的基本机制。 嗅觉电路提供了一个独特的模型,用于研究经验依赖的可塑性,部分原因是连续供应整合成人神经元。 我们的实验室最近实施了一个嗅觉提示学习范例,其中特定的气味对与奖励或惩罚相结合,以研究下游电路的变化。 以下协议概述了我们学习范式的基本设置。 在这里,我们描述设备设置,软件编程和行为训练方法。
【背景】成年大脑具有持续的经验依赖性结构变化。在首先处理气味信息的啮齿动物嗅球(OB)中,连续供应成年出生的中间神经元(颗粒细胞)或者整合到嗅觉电路中或经历凋亡(Petreanu和Alvarez-Buylla,2002; Carleton等,2003 ; Lledo等人,2006; Sakamoto等人,2014)。生存或死亡之间的选择受感觉刺激和嗅觉提示学习的影响很大(Rochefort等,2002; Alonso等,2006)。此外,较小的颗粒细胞在关键时间窗内也经历依赖于经验的突触变化(Yamaguchi and Mori,2005)。为了研究感觉体验如何影响OB电路中的突触可塑性,我们的实验室已经成功实施了Go / No-Go嗅觉学习任务(Huang et al。,2016; Quast et al。,2016)。训练小鼠将“气味”与水奖励和单独的“禁止气味”联系起来(试用超时)(图1)。完成训练后,小鼠将能够通过执行相关任务(精确度大于85%)来区分这两种气味(补充视频1)。

关键字:嗅觉, 电路, 学习, 突触, 可塑性, Go/No-Go, 行为

材料和试剂

  1. 由实验者选择的不同的气味对,以表示"Go"或S +刺激和"No-Go"或S-刺激。实施例:将1-丁醇和丙酸(分别为Sigma-Aldrich,目录号:437603和402907)稀释至10%的矿物油(Alfa Aesar,目录号:31911)(500μl矿物油中的气味剂)
  2. 带绿色聚丙烯孔盖的Qorpak硼硅酸盐玻璃小瓶(Qorpak,目录号:GLC-01016)
  3. Nalgene硅胶管(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:8600-0030)
  4. BD PrecisionGlide TM 18号斜角针(BD,目录号:305196)
  5. 成年小鼠(> 6周龄,我们的实验室平均使用12至19周龄的小鼠,平均体重为19g,女性为23g)

设备

  1. 超宽模块化测试室鼠标(Med Associates,目录号:ENV-307W)
  2. 不锈钢网格地板(Med Associates,目录号:ENV-307W-GFW)
  3. 针对宽鼠模块室的照明鼻响应(Med Associates,目录号:ENV-313W)(x2)
  4. 双通道嗅觉刺激(Med Associates,目录号:ENV-275)
  5. 照亮鼻子嗅觉响应与嗅觉口小鼠(Med Associates,目录号:ENV-375W-NPP)
  6. 独立USB接口,4输入/8输出仅与32位操作系统兼容(Med Associates,目录号:DIG-703A-USB)
  7. 带有Windows 2000,XP,Vista或7操作系统的标准台式电脑
  8. VWR流量计,压克力(VWR,目录号:97004-952)


    图2. Go/No-Go设备行为系统包含6个关键组件:行为室,水库,空气调节,USB接口系统,气味传递模块和个性化计算机(A)。室内空气首先从空气调节器传送到输入空气歧管,随后分配到两个含气味的玻璃小瓶和中心阀(B)。气味输出由两个螺线管(C)控制,可由Schedule Manager软件编程。气味进气管线,真空管路和两个分配器连接到行为室(D)。每个气味瓶与其自己的两个硅胶管配对,配有18号针头,以防止气味(E)的污染。

软件

  1. MED-PC IV行为软件套件(Med Associates,SOF-735)
    注意:此版本的软件现已被Med-PC V取代。
  2. 所有MED-PC用户(Med Associates,SOF-731)的MPC2XL数据传输实用程序,用于将数据重新格式化为Excel
  3. 计划管理软件(Med Associates,目录号:DIG-703A-USB),是编程培训阶段所需的

程序

  1. 建立行为室和程序化的嗅觉提示和奖励交付
    1. 获取并设置小鼠的操作性调理行为室
      1. 设置必须包括模块化的气味和输水口。
      2. 关联学习的编程培训阶段需要能够运行Med-PC IV软件的计算机。
      3. Med-PC IV软件使用专有的编程语言,实验者必须学习编程培训阶段。每个培训阶段的具体要求包括:
        1. 对于所有阶段:每个块的试验被阻止到20个试验以计算每个鼠标的每个块的性能。
        2. 第一阶段:程序必须指定当其鼻子捅入中心输水口时,鼠标会收到水报酬。此阶段的输出参数为:试用期限和收到的奖励数量。
        3. 阶段2:程序必须指定鼠标只有在将其鼻子插入气味递送口(中心端口)后才从两个侧面端口接收到水奖励。即使鼠标没有到达侧面端口进行奖励,中心戳之后应立即分配水。输出参数:试用期,试验次数和及时报酬次数(在中心港口的5秒钟内收到)。
        4. 第3阶段:程序必须指定向中心港口发送Go(S +)气味。只有在5秒钟的时间内,如果鼠标鼻子在侧端口发出气味,才应该分配水分。该程序还必须在中心端口鼻尖产生奖励所需的时间长度内以50毫秒逐步增加(从100-400毫秒)自动形成小鼠的行为。如果在20次试验的一个块内,鼠标在80%的时间内足够长的时间在中心端口上戳,程序应该将所需时间增加50毫秒。输出参数与第2阶段相同,加上收到的奖励数量和所有试验中正确答案百分比。
        5. 第3阶段:伪训练:该程序用于经过训练的小组对照组,而不考虑Go或No-Go(S-)气味的奖励或惩罚。所有参数与正常阶段3相同,除了不会引入S +气味。
        6. 第4A阶段:在这个阶段,程序必须从所有的试验开始,提供Go/S +加味剂,如阶段3.当小鼠完成1-2块Go/S +试验时, 85%的准确性,实验者可以对半数试验的S-/Go气味进行随机分配。如果鼠标不准确地在No-Go提示后寻求水奖励,则会给予2秒的超时罚款。为了帮助小鼠区分Go和No-Go试验,实验者可以在Go试验期间对端口附近的舱室进行编程,并在No-Go试验期间关闭。输出参数包括:试用时间,试验次数(中心口鼻孔数),完成试验次数(中心口鼻孔数超过400毫秒),正确响应数,接收到的奖励数。
        7. 第4B阶段:程序必须将老鼠的"超时"惩罚增加到4秒。输出参数与阶段4A相同。
        8. 第4阶段:伪训练:与第4B阶段相同,除了奖励和惩罚被随机分配给Go/S +和No-Go/S-气味。
        9. Go/No-Go:这个阶段代表"测试"阶段,并且可以在一对新颖的气味剂上进行,以测试如何精确地对实验对照小鼠进行气味关联。输出参数与阶段4A相同。如果在舞台4A中编程了侧灯,程序现在应该消除这些侧灯提示
  2. 关联学习(Go/No-Go)任务
    1. 小鼠的制备[3天]
      1. 在初步训练前3天,水被限制为40毫升/公斤/天,小鼠习惯于测试环境和室。
        1. 可以进行对对照动物的水限制的初步试验,以确定为维持行为任务的表现而提供的最小水量,而不超过人为的终点(National Research Council 2003),由实验者的IACUC指导确定。在我们手中,每只鼠每天40毫升/公斤足以维持性能,同时避免超过每只动物基线15%的体重减轻。这个终点是由于啮齿动物能够补偿逐渐的水分限制范例,并且能够承受高达基线体重的15%的损失,而不会显示遇险指标(Rowland,2007)
        2. 在限水期间,小鼠每天必须称重,以确保其高于基线质量的85%。如果小鼠低于该阈值,则从研究中移除,并提供自由水
        3. 请注意,与其他行为范例一样,结果可能会随着程序的细微变化而精细变化。因此,在培训开始之前,确保所有环境因素(温度,噪音,杂散气味)在整个协议中保持不变,实验观察者不会引入随机变异(例如,香水或观察员/处理程序的不规则变化)。
    2. 初步培训[8-24天]
      1. 小鼠分为控制组,伪训练组或训练组。
        1. 对照小鼠不再接受治疗。
        2. 伪训练小鼠的治疗方式与训练有素的组相同,除了每个试验均以相同的概率产生奖励和惩罚,无论所呈现的气味如何。
        3. 受过训练的小鼠进行Go/No-Go嗅觉关联学习范例。
      2. 配置行为室,只有一个中央端口可用于提供水。 (图3A)


        图3.行为室中的端口配置。阶段1包含中央水端口(A),侧面端口不可访问。所有其他阶段(包括Go/No-Go测试阶段)均采用中心气味口和两个水侧端口(B)进行。

      3. 对受过训练的训练有素的老鼠进行初步训练的以下阶段
        1. 阶段1 [1-3天,60分钟/小鼠/天]:小鼠学会将房间与水的存在联系起来。
          1)让每只老鼠探索房间,发现港口输送水。
          2)完成标准:60分钟内100次试验。
          注意:
          1)水阀的声音可能会使老鼠感到震惊,可能会停止演奏。如果老鼠太害怕探索,可以提供"自由"的水。这是通过使用软件上的定时输出选项来完成的,可以向输水口输送约200毫秒的水。
          2)如果尽管自由送水,如果鼠标冻结或停止探测,请让鼠标停留在腔室中,直到60分钟结束。删除并重复这个阶段在第二天的那只老鼠。
          3)在试验之间清空小鼠的固体废物。用70%乙醇喷洒并擦拭小室,以防止气味分散。
          4)即使在群体内,小鼠表现出自然的变异性。因此,当平均性能在上述标准的合理范围内(60分钟内完成100次试验)时,可以将小鼠作为一组升级为第2阶段。
        2. 阶段2 [1-5天,60分钟/小鼠/天]:小鼠被教导鼻捅入中心端口以获得从双方端口分配的自动水回报。
          1)配置室具有一个中心端口进行气味输送和两个侧面端口供水(图3B)。这将是实验剩余部分的房间。
          2)完成标准:40-50 + 60分钟试用,至少25-50%及时奖励。及时的奖励是在中心港口的鼻尖戳5秒内收到的。
        3. 阶段3 [1-3天,60分钟/小鼠/天]:小鼠必须先在5秒钟内进入中心端口,然后侧端口才能获得奖励。此外,在向任何一个侧面端口寻找水以获得分配的奖励之前,小鼠必须在中心突出更长时间(长达400毫秒)。这个阶段还介绍了Go/S +气味与水奖励配对。
          1)完成标准:> 60分钟内获得60奖励。
          2)对伪训练对照组应用3级伪训练,但不要引入S +/Go气味。
        4. 阶段4A [1-2天,60分钟/小鼠/天]:该阶段在Go vs. No-Go任务上训练小鼠,并介绍No-Go/S-气味,以及2秒的时间处罚小鼠在No-Go提示上寻求水奖励。该程序将从所有Go/S +气味演示开始。鼠标在所有Go/S +试验的1-2个块上表现良好后,参数可以切换到50%的Go/S +和50%的No-Go/S-试验。
          1)完成标准:40次试验与> 60%正确答案。
          2)对伪训练对照组应用4级伪训练
        5. 第4B阶段[5-11天,60分钟/小鼠/天]:这个阶段,在No-Go/S-试验中不正确地寻求奖励的老鼠处罚时间增加到4秒,或者没有在Go/S +审判。
          1)完成标准:> 60分钟内100次试验> 85%的准确度。
          注意:
          1)保持日常跟踪不仅每个会话的总百分比正确,而且每个会话的最大%和最小百分比是正确的,以监视每个鼠标在这个阶段的进度。
          2)在我们手中,85-90%的对照小鼠达到完成标准。如果研究要求小鼠继续进入Go/No Go测试阶段,我们建议排除不符合这些标准的老鼠,因为他们没有表现出足够的执行任务能力。 > 3)实验者可能希望在本阶段结束时对"反转"训练进行编程,其中Go和No-Go气味被置换在气味瓶平台上的位置。这是为了确保小鼠不会提示另一个刺激(例如, ,与一个或另一个端口相关联的特定阀的声音)。
    3. Go/No-Go试用[1天,〜20分钟/小鼠/天]
      1. 现在已经充分了解了训练阶段4B的小鼠已经充分了解了行为任务,并且可以暴露在新的气味对中,以测试他们区分新气味(例如,立体异构体如+和 - 柠檬烯)的能力。 。
      2. Go/No-Go试验的参数与第4B阶段的参数相同,除了中心端口300毫秒的鼻孔足以使老鼠执行Go或No-Go动作。
      3. 在程序中输入实验细节。将每只鼠标放入室内,并从屏幕界面中选择正确的程序运行。
      4. 统计分析的终点或变量由实验者确定,但在我们手中,控制小鼠通常会在大约200-400次试验或10-20次阻断之后显示新的气味对(> 85%正确反应)的准确性能。 br />

数据分析

在训练有素的组中,只有执行Go/No-Go试验的小鼠在>在我们的电路测绘实验中使用了85%的精度(Huang等人,2016)。不符合标准的小鼠不包括在进一步的研究中。此外,可以针对训练中使用的初始两种相关气味(图4A)以及任何后续重新相关联的气味(图4B)显示嗅觉学习进展。任何统计软件包都可用于分析数据,具体测试取决于实验者的需求。在Huang等人的中。 (2016),通过双因素方差分析比较了训练有素的,假性的和对照组中嗅球的连通性模式。

图4.学习曲线在第4B阶段,小鼠随机出现Go和No-Go试验。一组5只小鼠(A)显示了连续试验块的平均百分比正确选择(20次试验/阻断)。错误栏显示一组内所有动物的相同块的标准错误。一旦小鼠成功训练,重新结合新的气味剂也可以类似地绘制(B)。

笔记

  1. 为了减少实验之间的差异性,我们建议在所有测试组之间分配实验动物年龄和性别的对照组合。
  2. 确保行为室上的所有管道和配件都牢固地连接到其底部,以防止气味泄漏。
  3. 在低交通,昏暗的地方设置行为箱,以防止对小鼠的注意力。
  4. 保持室内空气流量为〜3-5L/min,并调节中心端口的真空吸力,以避免试验之间的气味。
  5. 使用气味专用管将气味小瓶连接到行为室。这样可以消除气味剂的交叉污染。
  6. 在笼子/套小鼠之间用70%EtOH彻底清洁盒子。

食谱

  1. 气味准备
    1. 在硼硅酸盐玻璃小瓶中加入500微升的气味和4.5毫升矿物油
    2. 准备两个1英尺长的硅管,并将其一端装入分开的18号针头。两针都将放入气味瓶中(补充视频2 ),管端将安装在进气口或出气口的气味盒上
    3. 总是在同一天准备新的气味。气味每周都会重塑

致谢

该协议是从我们实验室以前的工作(Huang等人,2016)中改编而成。它得到McNair医学研究所的支持,NINDS授予R01NS078294给B.R.A.和NIH IDDRC授予U54HD083092。作者声明没有利益冲突,没有竞争的利益。

参考

  1. Alonso,M.,Viollet,C.,Gabellec,MM,Meas-Yedid,V.,Olivo-Marin,JC和Lledo,PM(2006)。< a class ="ke-insertfile"href ="http: //www.ncbi.nlm.nih.gov/pubmed/17035535"target ="_ blank">嗅觉鉴别学习增加了嗅球中成人出生神经元的存活。 Neurosci 26(41):10508-10513。
  2. Carleton,A.,Petreanu,LT,Lansford,R.,Alvarez-Buylla,A.and Lledo,PM(2003)。< a class ="ke-insertfile"href ="http://www.ncbi。 nlm.nih.gov/pubmed/12704391"target ="_ blank">成为成年嗅球中的新神经元。 Nat Neurosci 6(5):507-518。 />
  3. Huang,L.,Ung,K.,Garcia,I.,Quast,KB,Cordiner,K.,Saggau,P.and Arenkiel,BR(2016)。< a class ="ke-insertfile"href = http://www.ncbi.nlm.nih.gov/pubmed/27559168"target ="_ blank">任务学习促进嗅球中的中间神经连通性图的可塑性。 Neurosci 36(34):8856-8871。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Liu, G., McClard, C., Tepe, B., Swanson, J., Pekarek, B., Panneerselvam, S. and Arenkiel, B. R. (2017). Olfactory Cued Learning Paradigm. Bio-protocol 7(9): e2251. DOI: 10.21769/BioProtoc.2251.
  2. Huang, L., Ung, K., Garcia, I., Quast, K. B., Cordiner, K., Saggau, P. and Arenkiel, B. R. (2016). Task learning promotes plasticity of interneuron connectivity maps in the olfactory bulb. J Neurosci 36(34): 8856-8871.
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