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Temporally-graded retrograde amnesia (TGRA) refers to a phenomenon of premorbid memory loss whereby information acquired recently is more impaired than information acquired more remotely. Studies of human amnesia have illuminated this phenomenon (Hodges, 1994; Squire and Alvarez, 1995), but such studies necessarily rely on retrospective methods and imperfect tests. Studies in experimental animals have the advantage that retrograde amnesia can be studied prospectively, the locus and extent of brain lesions can be determined accurately, and the timing and strength of original learning can be precisely controlled. The socially transmitted food preference (STFP) task has been one of the most productive rodent behavioral tasks to study TGRA (Clark et al., 2002).

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Socially Transmitted Food Preference (STFP) Task Protocol
可通过社会交往传播的食物偏好实验方案

神经科学 > 行为神经科学 > 学习和记忆
作者: Robert E. Clark
Robert E. ClarkAffiliation: Departments of Psychiatry, Neurosciences, and Psychology, University of California at San Diego (UCSD), La Jolla, USA
For correspondence: reclark@ucsd.edu
Bio-protocol author page: a53
Vol 2, Iss 11, 6/5/2012, 4586 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.224

[Abstract] Temporally-graded retrograde amnesia (TGRA) refers to a phenomenon of premorbid memory loss whereby information acquired recently is more impaired than information acquired more remotely. Studies of human amnesia have illuminated this phenomenon (Hodges, 1994; Squire and Alvarez, 1995), but such studies necessarily rely on retrospective methods and imperfect tests. Studies in experimental animals have the advantage that retrograde amnesia can be studied prospectively, the locus and extent of brain lesions can be determined accurately, and the timing and strength of original learning can be precisely controlled. The socially transmitted food preference (STFP) task has been one of the most productive rodent behavioral tasks to study TGRA (Clark et al., 2002).

[Abstract] 短暂性的逆行性遗忘(TFRA)是指依靠获取近期信息的暂时性失忆比依靠获取较久远的信息的暂时性失忆更有害的现象。人健忘症的研究已经解释了这一现象(Hodges, 1994; Squire and Alvarez, 1995), 但是这种研究必须依赖回忆法和不完全测试。利用实验动物来研究更加方便,因为逆行性遗忘可以被预测,脑损伤的位置和范围可以精确的确定,还可以精确的控制初始学习的时间和强度。 社会转交食物偏好(STFP)任务可以很好的研究啮齿类动物的暂时性逆行性失忆症TGRA(Clark et al., 2002)。

Materials and Reagents

  1. Rats
  2. Cinnamon
  3. Cocoa
  4. Ethanol
  5. Powder
  6. Flavored rat chow meal (see Recipes)

Equipment

  1. Glass jars and the jar holders
  2. Fisher Scientific scale
  3. Feeding apparatus
  4. Cages

Procedure

The Task:
The STFP task is separated into four distinct phases. Phase I consists of food deprivation and habituation of the demonstrator rats to the powder rat chow (“tuning of the demonstrator rats”). Phase II consists of the feeding of the demonstrator with a specified flavored meal, and the interaction of a demonstrator rat with an observer rat. Phase III is a specified delay interval following the interaction between the rats. Finally, phase IV is a testing period in which the observer rat is given a choice between a familiar (the flavored meal that the demonstrator rat was fed) and a novel (a flavor that neither rat has been exposed to) meal chow sample to test the memory of the interaction.

  1. Phase I: Food deprivation and habituation to meal chow
    In order to ensure that the demonstrator rats eat the desired flavored rat chow at the specified time, the rats must be habituation to the meal chow (finely ground rat chow pellets that the rats are normally fed). To do this the rats must become accustomed to a routine of 23 h of food deprivation, followed by one hour of feeding. This trains the rats to eat its fill within the hour ensuring the consistent transmission of the flavor from the demonstrator to the observer. The following steps are taken for this training.
    1. Day 1
      1. Cull the rats into individual cages so that all of the demonstrators and all of the observers are in their own cages.
      2. Remove the pellet rat chow from the demonstrators rats’ cages for 23 h, and place a “do not feed” sign on the cages.
    2. Day 2
      1. After the 23-h food deprivation, feed the demonstrator rats non-flavored meal chow for 1 h using the glass jars and the jar holders. Using a sharpie, label the glass jars with the rat number and the side (left or right) so the jars can be easily distinguished from each other.
      2. Using the Fisher Scientific scale, weigh the mass of one empty jar (massE) for each rat. Fill the jars approximately ¾ of the way full (45-50 g), and weigh the initial mass of each sample and jar (mass).
      3. Administer the samples to the rats using the jar holders in the rats' home cages.
        Place the sample holders and the samples in the demonstrator’s cage (opposite from the food and water bottle allowing for the maximum amount of room for the rat to eat) and bury the base of the holder in the bedding to stabilize the apparatus.
      4. After one hour has elapsed, remove the feeding apparatus and resume the 23-h food deprivation.
      5. Weigh the final mass of the sample and jar (massF) for each rat, and record the data.
    3. Day 3- same as day 2.
      NOTE: If a demonstrator rat fails to eat 5 g of powder chow during the one hour feeding session, he should not be used the following day in the interaction phase of the task. Additional days of training may be accrued until the rats reach the suggested criteria.
      1. After two days of 23 h food deprivation and one hour feeding, the rats should be accustomed to the eating schedule and the interaction with the observer rat will take place the following day.
      2. Using the large weigh boats, the two flavored meal chows should be prepared at this time. They are approximately 1% cinnamon (3 g of cinnamon to 300 g of meal chow) and 2% cocoa (6 g cocoa to 300 g of meal chow). Precautions should be taken so that cross contamination doesn’t occur during the preparation of the samples (i.e. change gloves after weighing out cinnamon or cocoa). Combine the meal and the given flavor in the same zip-lock bag and mix until evenly dispersed. It is recommended that the flavored meal should be prepared at least a day in advance and shouldn’t be stored for longer than a couple of days.

  2. Phase II: Interaction between the observer rats and the demonstrator rats.
    After the demonstrator rats have been trained to eat the meal chow, they will now be exposed to a flavored meal chow. This will be followed by an interaction with an observer rat allowing for the transmission of the scent of the specific flavor to the observer. The interaction is accomplished through a wire screen that is constructed in a cylindrical shape allowing the demonstrator rat to be placed into the empty space of the cylinder. This allows the rats to interact, the experimenter to differentiate between the two rats, and also prevents the rats from fighting.
    1. Day 4
      1. Weigh out the samples of the flavored powder chow, one jar each rat, (prepared the previous day) to be administered to the demonstrator rats. Record the empty mass of the jar (massE) and the initial mass of the sample (masso) on the data sheet.
        Note: It is important to always feed the same demonstrators the same flavor of meal. It is unclear if previous feedings of the demonstrator can influence or confuse the observer rats, therefore, feeding the demonstrators two different types of flavored meal chow for two different testing periods should always be avoided.
      2. After the 23-h food deprivation, feed the demonstrator rats flavored meal chow (cinnamon or cocoa) for 1 h.
      3. While the demonstrator rats are eating, remove the corresponding observer rats’ food.
      4. After the demonstrator rats have eaten the flavored powder chow for 1 h, place the cylindrical screen partition in the observer’s home cage and transfer the demonstrator rat into the center space of the screen partition. This separates the demonstrator and the observer rats. Replace the wire cage top to secure the partition and remove the water bottle. Allow the demonstrator and the observer rats to interact for the given exposure time. Typically 30 min, but this can be used as an independent variable.
        This is the only time that either rat is not allowed to have water present in their cages. It is unclear if drinking water can dilute the scent. It is also desirable to have the rats interacting as much as possible during this time so any distractions (e.g. a water bottle) should be avoided.
      5. While the demonstrator and the observer rats are interacting, determine the final mass of the demonstrator’s flavored powder chow samples and record the data.
      6. After the interaction time is complete, return the demonstrator rat to his home cage. It is easiest to remove both the rat and the screen partition concurrently since the demonstrator will often grab onto the screen once he is lifted out of the cage.
      7. If no further testing of the demonstrator rats will be done the following day, return the rat chow pellets and remove the do not feed sign from the demonstrator’s cage. If the demonstrators are going to be used in an interaction the following day, do not return their food.  Instead, continue with the 23 h. food deprivation schedule and return the caged demonstrator to the appropriate shelf.
      8. If a longer delay is used, replace the rat chow pellets and the water to the observer rat’s cage and return the caged observer to the shelf. Do not return the food if the observer rats are going to be tested immediately.  Instead, go directly to phase IV.

  3. Phase III: Delay interval.
    At this time, a given delay is allowed to elapse before the observer rats are given a choice between the food that their demonstrator rat was exposed to, the familiar food, and a novel flavored sample that neither rat has ever been expose to before. The length of this delay is variable and determined by the experimenter. This delay is crucial for post-operative animal recovery or to test the retention of the memory over longer delays.

  4. Phase IV: Testing phase of the observer rats.
    After the delay has elapsed, the observer rats are tested on the retention of the memory created during the interaction with the demonstrators. The test consists of exposing the observer rats to two samples, the familiar and the novel food samples, and determining by mass how much the observer rat eats of both samples. Measurements are made at one, two, and twenty four hour intervals and the percent preference for the familiar and novel food is calculated.
    1. Day 1
      1. After the given delay has elapsed, the observer rat’s food is removed and a “do not feed” sign is placed on the cage, one hour prior to administering the two samples.
      2. After removing the rat chow pellets, label two of the jars, weigh them empty (massE), and add the flavored rat chow meal  (1% cin and 2% cocoa) to approximately ¾ full (45-50 g) and determine the initial mass (masso).
      3. The two samples, novel and familiar, are taped (any kind will do) to the plastic sample holder and placed into a new fresh cage containing a minimal (just enough to cover the bottom of the cage) amount of bedding. The minimum bedding decreases the chances of the rat burying or contaminating the samples.  If a group of animals is being tested, alternate the familiar food on the left and the right sides so that an equal distribution between the two sides is obtained to compensate for any natural side preferences that the rats may have.
      4. Place the samples, one cinnamon and the other cocoa, into the new cage and move the observer rat, along with his water and wire cage top.
      5. Secure the cage and return it to the shelf, and allow the rat to eat from the two samples for one hour.
      6. After one hour has expired, both food samples are removed and weighed (massF1).
        Wipe away any bedding from the exterior of the jar and only attempt to remove contamination from the inside (by sifting) if there is an excess present.
      7. The samples are then returned to the test cage and the rat is allowed to eat for another hour.
      8. After the second hour, the samples are again removed, weighed, and the masses are recorded (massF2). The samples are placed back into the observers’ testing cage for another 22 h.
      9. After 24 h has elapsed, the samples and the do not feed sign are removed and the rat chow pellets are given back to the observer rat.
      10. The final samples are sifted (if necessary) through a screen to remove any bedding or other contaminating material and the final mass is measured (massF24).
        1. Use the small green handled sifting tool to sift away any contaminating material. It is easiest to sift the entire sample through the screen into a weigh boat and then transfer the entire sample back into the original glass container to make the final mass measurement.
        2. Often it is not necessary to sift the samples and any minor contamination can be removed using forceps. However, if there is contamination after the 24-hour exposure and if one sample of a pair is sifted, be sure to sift the other sample of the same pair. This ensures that if any of the samples mass is lost in the sifting process, it is proportionally lost in both samples so that the relative masses are roughly unchanged.

  5. Miscellaneous information
    1. Cleaning of glassware
    2. Organization of data
      All primary data should be kept in the black STFP binder. In addition to this, all data should be entered into the computer so that two copies of all the data are kept. The data sheets can be found in the Excel file under the C drive, Clark folder, Rat folder, and STFP folder. The data sheets used in the notebook are the “Data sheets for the STFP Task” and the data sheets used for the computer are the “Cal. data sheets for the STFP Task.” The calculation data sheets do the minor calculations needed to determine the mass consumed by the rats and can be used to save the experimenter time or as a check of any primary calculations. After all primary data has been recorded and stored; the data should be transferred to the “all data” sheet found in the same folder. This data file calculates both of the percent preferences and allows for quick and easy interpretation.
    3. Sample calculation
      Data is often interpreted as a percent preference for the familiar or novel food. This calculation is simply the mass of familiar/novel food consumed divided by the total mass of food eaten multiplied by 100%. Commonly, the percent preference for the familiar food is determined. For example,
                                       % preference =   mass of familiar food (g)      (100%)
                                                              Total mass of food eaten (g)

Recipes

  1. Flavored rat chow meal
    1% cinnamon
    2% cocoa

Acknowledgments

This protocol is adapted from Clark et al. (2002).

References

  1. Clark, R. E., Broadbent, N. J., Zola, S. M. and Squire, L. R. (2002). Anterograde amnesia and temporally graded retrograde amnesia for a nonspatial memory task after lesions of hippocampus and subiculum. J Neurosci 22(11): 4663-4669.

材料和试剂

 

1.         大鼠

2.         肉桂

3.         可可粉

4.         乙醇

 

实验设备

 

1.         玻璃罐

2.         Fisher天平

3.         饲喂器

 

实验步骤

 

任务:

STFP任务被分成四个不同的阶段。阶段一由实验鼠对粉末状鼠粮的厌恶和偏好组成。  阶段二由饲喂实验鼠特定风味的饲料和实验鼠和观察鼠之间的互作组成。阶段三是在鼠互相影响后有一个特定的延迟。最后,阶段四是一个测试期,让观察鼠正常饲料(实验鼠被饲喂的饲料)和新口味的饲料(实验鼠未接触过的饲料)中作做出选择以检测互作的记忆力。

1.        阶段一:食物的厌恶和偏好实验

为了确保实验鼠在特定时期吃到喜爱口味的鼠粮 .小鼠必需习惯这种鼠粮(小鼠经常被饲喂的精美颗粒鼠粮)要做到这一点,小鼠必需习惯吃食一小时后,禁食23小时。这样可以训练小鼠在一小时内吃饱保证实验鼠正确向观察鼠传递食物的风味。训练的步骤如下:

1)         第一天

a.         将小鼠分装在不同的笼子里,以确保实验鼠和观察鼠都在自己的笼子里。

b.        移走实验鼠笼子中的颗粒鼠粮23小时,并在笼子上放置禁止饲喂的标志牌。

2)         第二天

a.           经过23小时的禁食后,用玻璃罐和罐架饲喂实验鼠无味道的鼠粮一小时。将鼠号和方位标记在玻璃罐上以方便区分。

b.         用飞世儿天平称量每只小鼠的空玻璃罐的重量(mass E),罐子内大概装?满的样品(45-50 g),称量样品和罐子的初始总重量(masso)

c.         用罐架将样品提供给小时食用。

a)        将装有样品的架子放置在实验鼠的笼子中(方在饲料和饮水瓶的对面以保证鼠的采食空间),并在将架子的底部埋藏在笼子底部以固定架子。

d.        一个小时后,移走饲喂装置,继续禁食23小时。

e.         称量每只鼠的剩余样品和罐子的总重(massF)并记录数据。

3)         第三天,与第二天相同

注意:如果实验鼠在一小时的饲喂时间内吃的粉末饲料少于5g,那么它不能用于下面的互作实验。需要增加训练时间,直到它达到标准的要求为止。

a.         经过两天的23小时禁食,1小时吃食的训练后,小鼠需要按照安排吃食,与观察鼠的互作开始进行。

b.        这时需要准备两种口味的饲料。饲料中有大约1%的肉桂(每300g饲料添加3g肉桂)和2%的可可粉(每300g饲料添加6g可可粉)。需要准备备用样品,以防止准备样品时发生交叉污染(例如,称量肉桂或可可粉后要更换手套)。  将风味物质和饲料装在同样的自封袋内并进行混合直到它们均匀的分布在袋子内。建议风味饲料应该提前一天准备,并且不能保存不能超过2天。

2.        阶段二:观察鼠和实验鼠之间相互作用

在实验鼠被训练吃食后,它们可以吃风味食物了。然后实验鼠需要与观察鼠互作以传递饲料的特殊风味。互作是通过圆柱形金属丝网筛来实现的,实验鼠被放在圆柱体的空闲空间。这样可以保证小鼠互相接触又可以阻止小鼠相互打架。

1)         第四天

a.         称量粉末风味饲料的重量,每只鼠一个罐子,分配给实验鼠。在记录表上记录空罐子的重量(massE)和样品的初始重(masso)

注:饲喂同样的实验鼠同种风味的食物是非常重要的。并不知道先前实验鼠饲喂的饲料风味是否会影响或迷惑观察鼠,因此,避免在不同的测试期饲喂实验鼠两种不同风味的饲料。

b.        经过23小时的禁食后,饲喂实验鼠风味饲料(肉桂或可可粉)1小时。

c.         当实验鼠吃料时,移走相应观察鼠的食物。

d.        在实验鼠吃饲料1小时后,将圆柱形的金属丝网放置在观察鼠笼子中,并将实验鼠转移到金属丝网的中间。这样可以隔离实验鼠和观察鼠。将金属网的顶部移到原处保证隔离并移走饮水瓶。一般设置30分钟,但也可以将时间作为自变量。

a)        这段时间两种小鼠都不可以喝水,也是实验过程中唯一不能饮水的时间。因为并不知道饮水是否可以冲淡食物的气味。为了使这种小鼠充分的交流,可以在这段时间内移走所有的娱乐设施(如水瓶)。

e.         在实验鼠和观察鼠相互交流的过程中,可以称量实验鼠剩余的风味饲料的重量并记录。

f.          交流时间到后,将实验鼠移回自己的笼子。最简单的方法是将金属网和实验鼠一起移走,因为在将小鼠举起的过程中它会抓住金属网。

g.        如果不再进行试验,将实验鼠的颗粒饲料移回,并移走禁止饲喂的标志牌。如果接下来还要继续实验,就不需要移回它们的饲料。相反的要继续23小时的禁食训练,并将实验鼠笼转移到专用的架子上。

h.         如果开始进行时间延迟试验,那么将颗粒料和水瓶移回到观察鼠的笼子中,并将笼子放回专用的架子。如果观察鼠马上要做测试,那么不用移回食物,直接进入阶段四。

3.        阶段三:延迟时间

这一阶段,在观察鼠选择与实验鼠相同风味的食物和一种新风味的食物之前需要经过一段时间的延迟。延迟时间的长短是不同的,由实验者决定。延迟实验对手术后动物的恢复和检测长时间延迟后动物的记忆力是非常重要的。

4.        阶段四:测试观察鼠

延迟时间过后,检测观察鼠在与实验鼠接触过程中建立的记忆的保存。检测由两部分组成,一部分为让观察鼠接触两种饲料,与实验鼠相同的饲料和一种新口味的饲料;另一部分为称量观察鼠吃的这两种饲料的重量。称量在1小时、2小时和24小时进行,分别并计算两种饲料所占的百分比。

1)         第一天

a.          延迟时间过后,移走观察鼠的饲料,放置禁止饲喂的标志牌,一小时后提供两种口味的饲料。

b.        移走颗粒饲料后,将用来装两种饲料的罐子分别贴上标签,称量空罐子重(massE),将风味饲料(1%肉桂,和2%的可可粉)放入罐子?满处(45-50g),称量初始重(masso)

c.         两种饲料,新口味和正常口味,被捆在塑料样品架上,并放在一个有最少数量垫料(仅仅覆盖笼子的底部)的新笼子中。放置最低数值的垫料是为了减少观察鼠掩埋或污染饲料。如果有小鼠被测试,将同种饲料均匀分别在左右两边,以抵消小鼠习惯在某一边采食的偏好。

d.        将两种饲料放在新笼子中,并将观察鼠和金属网一起移入新笼子。

e.         关上笼子,放回原来的架子上,保证小鼠采食两种饲料1小时。

f.          一小时后,移出两种饲料并称重(massF1)

a)        清理罐子表面的垫料,如果罐子中有过多的污染物,将其移出(用过滤的方法)。

g.        将饲料重新放进笼子让小鼠再吃1小时。

h.         第二个1小时过后,移出饲料,称重(massF2)。称重后饲料放回笼子中22小时。

i.           小鼠一共采食24小时后,移走样品饲料和禁止饲喂的标志牌,移回颗粒饲料。

j.           用筛子过滤剩余的饲料(如果有必要),除去垫料和其他污染物,称量最后的重量(massF24)

a)        用小的绿色把柄的筛子过滤掉污染物。可以将全部饲料筛到称量盘中然后再将全部饲料倒回原来的罐子中称重。

b)        通常不必过滤饲料,可以用镊子移走小的污染物。然而,24小时后通常有较多的污染物,如果过滤其中一个罐子中的饲料,那么另外一个配对的罐子也要过滤。这样可以保证如果筛的过程中有饲料损失,那么每一个罐子损失的比例相同。

5.        相关信息

1)         清洗玻璃器皿

a.         所有的玻璃器皿和塑料样品架都要彻底的用玻璃清洗机清洗,并用温的或热的自来水漂洗,然后用蒸馏水洗3次。玻璃罐在装样品前用95%的乙醇擦拭。这些步骤可以降低器皿残留气味在饲喂和测试过程中的影响。

2)         数据整理

a.         所有的原始数据保存在黑色的STFP活页夹中。另外,所有的数据需要输入电脑,以保证所有数据有两份拷贝。数据以excel的形式保存在C/Clark文件夹/Rat文件夹/STFP文件夹。笔记本中的数据表是“Cal. data sheets for the STFP Task.”,电脑中的数据表是“Cal. data sheets for the STFP Task.”。计算数据表可以做较简单的计算如计算小鼠消耗的饲料质量,可以用来节省实验时间或是用来检查原始数据。所有的原始数据被记录和保存后,数据需要转换成“all data”工作做表保存在相同的文件夹。这个数据文件包括当前偏好和快的和简单的注释。

3)         样品计算

a.         数据计算成对正常口味或新口味饲料的偏好百分比。计算很简单,为小鼠吃的正常口味/新口味饲料占总消耗饲料的百分比。通常计算正常饲料的偏好百分比。例如:

%偏好=[正常口味饲料重量(g/总采食饲料质量(g]×100%

 

参考文献

 

1.         Clark R.E., Broadbent N.J., Zola S.M., Squire L.R. (2002). Anterograde amnesia and temporally graded retrograde amnesia for a nonspatial memory task after lesions of hippocampus and subiculum. Journal of Neuroscience 22(11): 4663-9. 

 

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How to cite this protocol: Clark, R. E. (2012). Socially Transmitted Food Preference (STFP) Task Protocol. Bio-protocol 2(11): e224. DOI: 10.21769/BioProtoc.224; Full Text



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