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Context-driven Salt Seeking Test (Rats)
情景驱动的大鼠寻盐测试   

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The Journal of Neuroscience
Jun 2017

Abstract

Changes in reward seeking behavior often occur through incremental learning based on the difference between what is expected and what actually happens. Behavioral flexibility of this sort requires experience with rewards as better or worse than expected. However, there are some instances in which behavior can change through non-incremental learning, which requires no further experience with an outcome. Such an example of non-incremental learning is the salt appetite phenomenon. In this case, animals such as rats will immediately seek out a highly-concentrated salt solution that was previously undesired when they are put in a novel state of sodium deprivation. Importantly, this adaptive salt-seeking behavior occurs despite the fact that the rats never tasted salt in the depleted state, and therefore never tasted it as a highly desirable reward.

The following protocol is a method to investigate the neural circuitry mediating adaptive salt seeking using a conditioned place preference (CPP) procedure. The procedure is designed to provide an opportunity to discover possible dissociations between the neural circuitry mediating salt seeking and salt consumption to replenish the bodily deficit after sodium depletion. Additionally, this procedure is amenable to incorporating a number of neurobiological techniques for studying the brain basis of this behavior.

Keywords: Salt appetite (盐嗜好), Conditioned place preference (条件性位置偏好)

Background

The salt appetite phenomenon was first discovered by Richter (1936), who found that rats began to immediately consume a 1% salt solution in greater quantities compared to water in a 2-bottle choice test following adrenalectomy and consequently bodily sodium depletion. More recently, Robinson and Berridge (2013) have shown that this immediate increase in salt-seeking behavior occurs to discrete salt-paired cues following sodium depletion in the absence of salt before it has been tasted as a desirable reward. In addition, adaptive salt-seeking has also been observed with contextual cues following sodium depletion (Stouffer and White, 2005).

In the brain, there is clear evidence that the central nucleus of the amygdala (Galaverna et al., 1993; Seeley et al., 1993; Tandon et al., 2012; Hu et al., 2015), lateral hypothalamus (Wolf and Quartermain, 1967; Tandon et al., 2012), and the nucleus accumbens (Roitman et al., 2002; Voorhies and Bernstein, 2006; Loriaux et al., 2011; Tandon et al., 2012) are important for the consumption of salt following sodium depletion in order for animals to replenish the deficit. However, there has been surprisingly little work done on the neural circuitry mediating cue-driven salt seeking following sodium depletion. In other words, it is mostly unclear how the brain enables animals to seek out salt in a novel deprivation state. In a recent study, we showed that the ventral pallidum (VP) plays an important role in this phenomenon (Chang et al., 2017). The VP has previously been shown to track the changes in value of salt-paired cues before and after sodium depletion (Tindell et al., 2009; Robinson and Berridge, 2013). However, it was previously unknown whether the VP is necessary for mediating salt appetite in terms of cue-driven salt seeking or salt consumption. Using a novel CPP procedure, described here, we showed that optogenetic inhibition of the VP impairs context-driven salt seeking but not the consumption of salt itself following sodium depletion (Chang et al., 2017).

The protocol we have used to demonstrate this effect allows for not only optogenetics to be used but also other techniques to manipulate the brain (e.g., DREADDs, intracranial injections, lesions) or to record brain activity (e.g., electrophysiology, calcium imaging). Further study of context-driven salt seeking with this procedure may help elucidate the neural bases of disorders of aberrant motivation that may lead to reduced reward seeking, as in depression, or non-homeostatic reward seeking (e.g., overeating leading to obesity). In addition, this procedure could be easily extended to investigate the neural bases of other nutrient deficit-induced changes in behavior such as calcium appetite (Leshem et al., 1999; Schulkin, 2000).

Materials and Reagents

  1. Electrical tape (TemflexTM 1700, 3M, catalog number: 1700-1X66FT )
  2. Disposable weighing boat
  3. Rat (Long-Evans, 250-300 g; Male; 7 weeks old; Charles River Laboratories)
  4. Sodium-free food (TestDiet; Order Info: 1816123 (5ANR), TD 90228 1/2)
  5. Sugar (pure cane granulated, Domino®)
  6. Salt (iodized, Morton Salt, Inc.)
  7. Sugar-free Kool-Aid powder (orange and grape, Kool-Aid, Kraft Foods, Inc.)
  8. Furosemide (Salix®, Merck)

Equipment

  1. Preference test chamber (29.5 x 12.5 x 21 in., custom designed; Dartmouth Apparatus Shop)
  2. 16 oz. screw-top bottles (Ancare, catalog number: MST16 )
  3. Screw-top bend ball pt. tubes (5 in. long with 1 in. bend, Ancare, catalog number: PCST51BTD )
  4. Digital video camera (Sony)
  5. 2 portable luminaires (UL, catalog number: E196460 )
  6. 2 red light bulbs (Sunlite, catalog number: SL24/R )

Procedure

  1. Preparation of animals
    This procedure is designed for rats as the subjects. Rats are food restricted and maintained at 85% of their ad libitum weights throughout the experiment to motivate them to drink the sucrose and salt solutions.

  2. Preparation of apparatus
    1. Behavioral training is conducted in a custom-designed place preference chamber (29.5 x 12.5 x 21 in.), which we had made by the Dartmouth Apparatus Shop (Figure 1). The outside walls of the chamber are made of transparent acrylic, and there are acrylic inserts that can be placed in the chamber to divide it into three separate contexts. All training and testing is conducted in the dark except for the red light provided by the portable luminaires (see Figure 1) to provide enough illumination for recording purposes.


      Figure 1. Setup for investigating context-driven salt seeking and close up views of the place preference chamber

    2. Two contexts (12.5 x 12.5 x 18.5 in.) within the chamber are paired with one of two solutions, while the third middle context (5 x 12.5 x 18.5 in.) connects the other two contexts and is paired with nothing. One of the paired contexts is designated the Grid context. The Grid context has a transparent acrylic floor in a grid-like pattern with electrical tape on the outside walls that form a grid-like pattern. The other paired context is designated the Striped context. The Striped context has the same grid-patterned floor, but the outside walls have electrical tape in a parallel diagonal pattern. Each of these two contexts also has a distinctive object to aid in discrimination (e.g., a metal binder clip or an empty plastic microscope coverslip case). Both contexts have holes on either end to allow for a spout connected to a bottle filled with a solution to be available for the rats to drink from.
    3. The third, middle context that connects the Grid and Striped contexts is designated the Neutral context that consists only of clear acrylic walls and is the starting point for each training and testing session. Although individual rats can sometimes naturally favor one context, it is important to first establish that there is no general bias towards one context of the other. If there is, adjustments can be made (e.g., to the lighting, chamber orientation, etc.).

  3. Behavioral training and testing protocol
    Step 1: Training
    1. Rats undergo 8 days of initial training in which they learn to associate one context (Grid or Striped) with access to 3% sucrose in distilled water, and the other context with access to 3% salt in distilled water. Each solution is mixed with 2 g of sugar-free Kool-Aid powder (orange or grape) to aid discrimination between the two contexts and solutions. The assignment of context, solution, and Kool-Aid flavor pairings are counterbalanced across rats but kept constant within each rat.
    2. On each training day (4 days total in each context), rats are placed into the Neutral context and allowed to freely explore one context (Grid or Striped) for 20 min. Access to the other paired context is blocked. During these sessions, rats are allowed to freely drink from a bottle of 100 ml of either sucrose or salt (orange and purple bottles as in Step 1 of Figure 2). The order of context exposure is pseudorandomly assigned between rats.


      Figure 2. Behavioral training and testing procedure to investigate context-driven salt seeking in rats. Step 1: Rats are trained over 8 days to associate one context with access to sucrose (e.g., Grid) and another context with access to salt (e.g., Striped). Step 2: Rats are then given a baseline test session in which they have access to both contexts without sucrose or salt (but flavored solutions still present). Following 4 days of retraining (2 days in each context), rats are then depleted of bodily sodium through furosemide injections (i.p.). Step 3: After 48 h, rats are placed back into the chamber for a depletion test that is identical to the baseline test session. Step 4: Finally, rats are given a consumption test that is identical to the previous test sessions but sucrose and salt are now available. All training and testing sessions are 20 min in length.

    Step 2: Baseline test
    1. Once initial training is complete, rats are then given a 20 min baseline preference test in which they are allowed to explore the entire chamber, including both the Grid and Striped contexts, for the first time (both wall inserts are removed). The Grid and Striped contexts in this test session are identical as in training. One important exception is that sucrose and salt are not present in the flavored solutions. We conduct the baseline preference test in this way to gauge rats’ ability to use the contextual cues of the chamber to guide their behavior. The test serves as an important comparison point for an identical test conducted after sodium depletion.

    Retraining and sodium depletion
    1. Following the baseline test session, the rats are given an additional 4 days of training (2 sessions in each context) that is identical to the initial training sessions (Step 1). We give rats an additional 4 days of training to re-strengthen the associations between sucrose and salt with their respective contexts, which may be weakened following the baseline preference test. After the final training session, rats are given systemic injections of the diuretic furosemide (10 mg/ml/kg; i.p.; Merck). In order to maintain sodium deprivation, rats are also maintained on a sodium-free diet (TestDiet) and distilled water immediately following furosemide injections.

    Step 3: Depletion test
    1. Forty-eight hours after the furosemide injection, rats are then given another context preference test (depletion test). This test is identical to the baseline preference test (i.e., the full chamber is accessible; flavored solutions are available, but contain no salt or sucrose), except now the rats are sodium-deprived. We perform the depletion test this way in order to directly compare the elevation in time spent in the salt-paired context following sodium depletion compared to the baseline preference test.

    Step 4: Consumption test
    1. The next day, rats are given a final context preference test with sucrose and salt now present in the flavored solutions (consumption test). This allows us the opportunity to confirm that rats are indeed sodium-depleted. It also further provides an opportunity to investigate any possible neural dissociations between context-driven salt seeking (depletion test) and the consumption of salt (consumption test) following sodium depletion. Upon completion of this test, rats are placed back on their normal food and water diet.

Data analysis

  1. Video of test sessions is recorded by a digital camera (Sony) that is attached to a tripod facing the side of the chamber and positioned so that the entire chamber is visible. The primary measures to analyze are the amount of solutions consumed (in ml) during training and test sessions, the number of entries into each paired context during test sessions, the amount of time spent in each paired context (in sec) during test sessions, and the amount of time spent consuming solutions (in sec) during test sessions. The amount of time spent in each context and the amount of time spent consuming solutions is also broken down into 5-min blocks to give a more detailed record of how rats’ behavior changes over time. Behavior is hand-scored offline by looking at the head placement of each rat and what context it is located in, calculated to the nearest second. Additionally, calculations of elevation scores of each measure can be made by computing the total difference between depletion and baseline tests. Automated video-tracking devices can be used as an alternative to video hand-scoring. Spout lickometers can also be incorporated to assess lick microstructure in addition to volume consumption.
  2. For analyses with multiple data points from each subject (e.g., training consumption data or 5-min block data), repeated measures ANOVAs are used. For analyses with a single data point from each subject (e.g., elevation scores) based on a priori test plans, generalized linear models can be used. Significant interactions are followed up with Bonferroni-corrected generalized linear models. Otherwise, all analyses have a rejection criterion of P < 0.05.
  3. Figure 3 presents what one would expect to observe for a control group of rats in our salt appetite procedure (n = 19 male rats; group means ± SEM; adapted from Chang et al., 2017). Rats should drink more sucrose than salt over the course of training (Figure 3A). Following sodium depletion, rats should show an elevation in time spent in the salt paired context during the depletion test compared to the baseline test (Figure 3B). Finally, rats should show in elevation in the consumption of salt and a decrease in the consumption of sucrose compared to the last training day with each solution following sodium depletion (Figure 3C). Neural manipulations during either the depletion and consumption tests may reveal dissociations between context-driven salt seeking and salt consumption following sodium depletion. One salient example is our prior study (Chang et al., 2017), in which we observed a specific deficit in context-driven salt seeking but not salt consumption following sodium depletion with optogenetic inhibition of the VP.


    Figure 3. Expected results with our salt appetite procedure. A. Rats should drink more sucrose than salt over the course of training. B. Rats should show an elevation in time spent in the salt-paired context and a decrease in time spent in the sucrose-paired context following sodium depletion during the depletion test compared to the baseline test. C. Rats should show an elevation in the consumption of salt and a decrease in the consumption of sucrose compared to the last training day with each solution following sodium depletion. Figures adapted from Chang et al., 2017.

Notes

  1. Although we use ball pt. tubes, there may still be some leaking of solutions when placing bottles into position before the start of each session. To minimize the amount of solution lost from leaking, we place a collection dish (e.g., a disposable weighing boat) underneath the spout on the floor of the chamber to collect any solution that may leak before the rat is placed in the chamber. Once the bottle has been in position for approximately 5 min, the collection dish is then removed before the rat is placed into the chamber. The solution in the collecting dish is then added to what is remaining in the bottle following each session to provide a more accurate measurement of how much rats actually consumed during each session.
  2. Some individual rats may show a bias towards one context of the chamber in terms of time spent in each context regardless of which solution it is paired with during the baseline test session. However, what is most critical is the elevation in time spent in each context during the depletion test that is the primary measure for gauging the salt appetite phenomenon. We have consistently observed a robust elevation in time spent in the salt-paired context following sodium depletion in our control rats using this method of measurement despite any inherent biases rats may have towards one context or the other.
  3. We have only run this procedure using male rats, but we would not expect to see differences between male and female rats.

Recipes

  1. For training sessions, we make each solution in 1 L batches. Thus, 30 g of either sugar or salt are weighed out and placed into a 1 L bottle. The bottle is then filled up to 1 L with distilled water. Finally, 2 g of sugar-free Kool-Aid powder (orange or grape) is added. The solution is then mixed until either the sugar or salt (and Kool-Aid powder) is dissolved.
  2. For test sessions, 1 L batches are filled with distilled water and 2 g of Kool-Aid powder only.
  3. Furosemide is initially at a 5% concentration, which we dilute it to 1% with distilled water (e.g., 5 ml of furosemide and 20 ml of distilled water) for a concentration of 10 mg/ml.

Acknowledgments

This work was supported by funding from National Institutes of Health Grant F32MH106178 (SEC) and from Whitehall Foundation Research Grant 2014-05-77 (KSS). The authors declare no competing financial interests. This protocol was adapted from Chang et al., 2017.

References

  1. Chang, S. E., Smedley, E. B., Stansfield, K. J., Stott, J. J. and Smith, K. S. (2017). Optogenetic inhibition of ventral pallidum neurons impairs context-driven salt seeking. J Neurosci 37(23): 5670-5680.
  2. Galaverna, O. G., Seeley, R. J., Berridge, K. C., Grill, H. J., Epstein, A. N. and Schulkin, J. (1993). Lesions of the central nucleus of the amygdala. I: Effects on taste reactivity, taste aversion learning and sodium appetite. Behav Brain Res 59(1-2): 11-17.
  3. Hu, B., Qiao, H., Sun, B., Jia, R., Fan, Y., Wang, N., Lu, B. and Yan, J. Q. (2015). AT1 receptor blockade in the central nucleus of the amygdala attenuates the effects of muscimol on sodium and water intake. Neuroscience 307: 302-310.
  4. Leshem, M., Del Canho, S. and Schulkin, J. (1999). Calcium hunger in the parathyroidectomized rat is specific. Physiol Behav 67(4): 555-559.
  5. Loriaux, A. L., Roitman, J. D. and Roitman, M. F. (2011). Nucleus accumbens shell, but not core, tracks motivational value of salt. J Neurophysiol 106(3): 1537-1544.
  6. Richter, C. P. (1936). Increased salt appetite in adrenalectomized rats. Am J Physiol 115: 155-161.
  7. Robinson, M. J. and Berridge, K. C. (2013). Instant transformation of learned repulsion into motivational "wanting". Curr Biol 23(4): 282-289.
  8. Roitman, M. F., Na, E., Anderson, G., Jones, T. A. and Bernstein, I. L. (2002). Induction of a salt appetite alters dendritic morphology in nucleus accumbens and sensitizes rats to amphetamine. J Neurosci 22(11): RC225.
  9. Schulkin, J. (2000). Calcium hunger: Behavioral and biological regulation. Cambridge University Press.
  10. Seeley, R. J., Galaverna, O., Schulkin, J., Epstein, A. N. and Grill, H. J. (1993). Lesions of the central nucleus of the amygdala. II: Effects on intraoral NaCl intake. Behav Brain Res 59(1-2): 19-25.
  11.  Stouffer, E. M. and White, N. M. (2005). A latent cue preference based on sodium depletion in rats. Learn Mem 12(6): 549-552.
  12. Tandon, S., Simon, S. A. and Nicolelis, M. A. (2012). Appetitive changes during salt deprivation are paralleled by widespread neuronal adaptations in nucleus accumbens, lateral hypothalamus, and central amygdala. J Neurophysiol 108(4): 1089-1105.
  13. Tindell, A. J., Smith, K. S., Berridge, K. C. and Aldridge, J. W. (2009). Dynamic computation of incentive salience: "wanting" what was never "liked". J Neurosci 29(39): 12220-12228.
  14. Voorhies, A. C. and Bernstein, I. L. (2006). Induction and expression of salt appetite: effects on Fos expression in nucleus accumbens. Behav Brain Res 172(1): 90-96.
  15. Wolf, G. and Quartermain, D. (1967). Sodium chloride intake of adrenalectomized rats with lateral hypothalamic lesions. Am J Physiol 212(1): 113-118.

简介

基于预期和实际发生的差异,通过增量学习经常发生奖励寻求行为的变化。这种行为灵活性要求奖励的经验比预期更好或更差。然而,在一些情况下,行为可以通过非增量学习来改变,这不需要进一步的经验与结果。这种非增量学习的例子是盐食欲现象。在这种情况下,像老鼠这样的动物会立即寻找高浓度的盐溶液,这种盐溶液在处于新的钠剥夺状态时以前是不受欢迎的。重要的是,这种适应性寻盐行为发生,尽管大鼠从未在耗尽状态下尝过盐,因此从来没有尝到过它是一种非常理想的奖励。

以下协议是调查神经电路介导自适应寻盐使用条件位置偏好(CPP)程序的方法。该程序旨在提供发现神经回路介导寻盐和盐消耗以补充缺钠后身体缺陷之间可能的解离的机会。另外,这个程序适合于结合许多神经生物学技术来研究这种行为的大脑基础。

【背景】盐食欲现象最早是由里希特(Richter,1936)发现的,他发现大鼠开始立即食用1%的盐溶液,与肾上腺切除术后的2瓶选择试验中的水相比,其量更大,因此体内钠耗竭。最近,Robinson和Berridge(2013)已经表明,在没有盐的情况下钠消耗之前,这种盐寻求行为的立即增加发生在离散的盐配对线索之后,因为它已被尝试作为理想的奖励。此外,在钠耗竭后,情景线索也观察到了适应性寻盐(Stouffer和White,2005)。

在大脑中,有明确的证据表明杏仁核的中心核(Galaverna et al。1993; Seeley et al。1993; Tandon et al。 ,2012; Hu等人,2015),侧下丘脑(Wolf and Quartermain,1967; Tandon等人,2012)和细胞核(Roitman等人,2002; Voorhies和Bernstein,2006; Loriaux等人,2011; Tandon等人,2012)对钠消耗后食用盐是重要的,以便动物补充赤字。然而,神经电路在钠耗竭后介导提示驱动盐寻找方面做了令人惊讶的少量工作。换句话说,大脑如何使动物在新的剥夺状态下寻找盐是很不清楚的。在最近的一项研究中,我们发现腹侧苍白球(VP)在这种现象中起着重要作用(Chang等人,2017)。先前已证明,VP在钠耗竭前后跟踪盐配对线索值的变化(Tindell et al。,2009; Robinson and Berridge,2013)。然而,之前还不知道VP是否需要在提示驱盐或盐消耗方面调解盐的食欲。使用这里描述的一种新颖的CPP程序,我们发现VP的光遗传学抑制损害上下文驱动的寻盐,但不会消耗钠盐本身(Chang等人,2017)。

我们用来证明这种效应的协议不仅允许使用光遗传学,而且还可以使用其他技术来操纵大脑(例如,DREADDs,颅内注射,病变)或记录大脑活动( 例如,电生理学,钙成像)。进一步研究上下文驱动的盐寻求这一过程可能有助于阐明异常动机障碍的神经基础,这可能导致减少寻求奖励,如抑郁症或非稳态奖励寻求( eg ,暴饮暴食导致肥胖)。此外,这个程序可以很容易地扩展到调查其他营养缺乏诱导的行为变化如钙食欲的神经基础(Leshem等人,1999; Schulkin,2000)。

关键字:盐嗜好, 条件性位置偏好

材料和试剂

  1. 电工胶带(Temflex TM 1700,3M,目录号:1700-1X66FT)
  2. 一次性称量船
  3. 大鼠(Long-Evans,250-300g;雄性; 7周龄; Charles River Laboratories)
  4. 无钠食品(TestDiet;订单信息:1816123(5ANR),TD 90228 1/2)
  5. 糖(纯蔗糖粒,Domino )
  6. 盐(加碘,莫顿盐公司)

  7. 无糖Kool-Aid粉(橙和葡萄,Kool-Aid,卡夫食品公司)
  8. 速尿(Salix ,Merck)

设备

  1. 偏好测试室(29.5 x 12.5 x 21英寸,定制设计;达特茅斯仪器店)
  2. 16盎司。螺杆瓶(Ancare,产品目录号:MST16)
  3. 螺旋顶部弯曲球pt。 (5英寸长,1英寸弯头,Ancare,目录号:PCST51BTD)
  4. 数码摄像机(索尼)
  5. 2个便携式灯具(UL,目录号:E196460)
  6. 2个红色灯泡(Sunlite,产品目录号:SL24 / R)

程序

  1. 准备动物
    这个程序是为老鼠设计的。在整个实验过程中,大鼠的食物都受到限制并保持在其自由采食量的85%,以激励他们饮用蔗糖和盐溶液。

  2. 仪器的准备
    1. 行为训练是在达特茅斯器械商店(图1)制作的定制设计的地点偏好室(29.5 x 12.5 x 21英寸)中进行的。腔室的外壁由透明的丙烯酸树脂制成,并且可以放置在腔室中的丙烯酸插入件将其分成三个独立的环境。除了便携式灯具提供的红光(见图1)以外,所有的培训和测试均在黑暗中进行,以便为记录目的提供足够的照明。


      图1.调查环境驱动的寻盐和关闭地点偏好室的视图的设置

    2. 腔体内的两个环境(12.5 x 12.5 x 18.5英寸)与两个解决方案中的一个配对,而第三个中间环境(5 x 12. 5 x 18.5英寸)连接另外两个环境并且没有配对。配对的上下文之一被指定为网格上下文。网格环境中有一个透明的丙烯酸地板,网格状图案,外壁上有电气胶带,形成网格状图案。另一个配对上下文被指定为条纹上下文。条纹背景具有相同的网格图案的地板,但外墙具有平行对角线图案的电磁带。这两种情境中的每一种都有一个区别对象来帮助辨别(例如金属活页夹或空的塑料显微镜盖玻片情况)。两种情况在两端都有漏洞,以允许连接到充满解决方案的瓶子的喷口供老鼠饮用。
    3. 连接网格和条纹上下文的第三个中间上下文被指定为仅包含透明丙烯酸墙壁的中性上下文,并且是每个培训和测试会话的起点。虽然个体老鼠有时候自然会喜欢上下文,但重要的是首先确定对另一个上下文没有普遍的偏见。如果有的话,可以进行调整(例如,照明,房间方向,等等)。

  3. 行为训练和测试协议
    第1步:培训
    1. 大鼠进行为期8天的初始训练,其中他们学习将一种情况(格栅或条纹)与3%蔗糖在蒸馏水中的接触联系起来,另一种情况下接触蒸馏水中3%的盐。每种溶液都混有2克无糖Kool-Aid粉(橙或葡萄),以帮助区分两种情况和解决方案。上下文,解决方案和Kool-Aid风味配对的分配在大鼠之间是平衡的,但在每只大鼠中保持不变。
    2. 在每个训练日(每种情况下总共4天)中,将大鼠置于中性背景中并允许自由探索一个背景(网格或条纹)20分钟。访问其他配对的上下文被阻止。在这些过程中,允许大鼠从一瓶100ml的蔗糖或盐(如图2的步骤1中的橙色和紫色瓶)中自由饮用。上下文暴露的顺序是伪随机分配给老鼠的。


      图2.行为训练和测试程序,用于研究上下文驱动的大鼠寻盐过程。第一步:大鼠接受8天的培训,将一种情况与获得蔗糖联系起来( >,Grid)和另一个可以访问salt的上下文(,例如,Striped)。步骤2:然后给大鼠一个基线测试期,在这个测试期中,他们可以在没有蔗糖或盐的情况下使用这两种情况(但仍然存在有味的溶液)。经过4天的再训练(在每种情况下2天)后,通过速尿(i.p.)给大鼠排出体内钠。步骤3:48小时后,将大鼠放回腔室进行与基线测试阶段相同的耗尽测试。步骤4:最后,给大鼠进行与之前的测试相同的消费测试,但现在可以获得蔗糖和盐。所有的培训和测试都是20分钟。

    第2步:基线测试
    1. 一旦初始训练完成,大鼠接着进行20分钟的基线首选测试,其中允许他们首次探测整个腔室(包括网格和条纹背景)(两个壁插入物都被移除)。本次测试中的网格和条纹上下文与训练中的相同。一个重要的例外是蔗糖和盐不存在于调味溶液中。我们以这种方式进行基线偏好测试,以测量老鼠使用房间的情境提示来指导他们的行为的能力。该测试作为钠耗竭后进行的相同测试的重要比较点。

    再培训和钠耗竭
    1. 在基线测试阶段之后,对老鼠进行另外4天的训练(在每种情况下2次训练),这与初始训练阶段相同(步骤1)。我们给予老鼠额外4天的训练,以重新加强蔗糖和盐之间的关联,并且各自的情况可能会在基线偏好测试后减弱。在最后一次训练之后,给大鼠全身注射利尿速尿(10mg / ml / kg; i.p。; Merck)。为了维持钠剥夺,在注射呋塞米后,大鼠也立即保持无钠饮食(TestDiet)和蒸馏水。

    第3步:耗尽测试
    1. 呋塞米注射后48小时,然后给予大鼠另一情境偏好测试(耗竭测试)。该测试与基线偏好测试相同(即,完整的室可用;有风味的溶液可用,但不含盐或蔗糖),除了现在大鼠钠剥夺之外。我们通过这种方式进行耗竭测试,以便直接比较钠耗竭后在盐配对环境中花费的时间与基线偏好测试相比所花费的时间。

    第4步:消费测试
    1. 第二天,给予大鼠蔗糖和现在存在于调味溶液中的盐(消耗测试)的最终上下文偏好测试。这使我们有机会证实大鼠确实缺钠。它还进一步提供了一个机会,可以研究在耗尽钠后,上下文驱动的寻盐(耗竭测试)和盐消耗(消耗测试)之间任何可能的神经解离。在完成这个测试后,大鼠被放回正常的食物和饮水。

数据分析

  1. 测试会议的视频由数码相机(索尼)记录,该数码相机安装在面向室内一侧的三脚架上并定位,以便可以看到整个室。要分析的主要措施是在培训和测试会话期间消耗的解决方案数量(单位为毫升),测试会话期间每个配对上下文的条目数量,测试期间每个配对上下文(以秒为单位)花费的时间量,以及在测试会话期间花费解决方案的时间(以秒为单位)。每种情况下消耗的时间量和消耗解决方案的时间量也被分解为5分钟的块,以更详细地记录老鼠的行为随时间变化的情况。行为是通过查看每只老鼠的头部位置以及它所处的位置,以最近的秒计算的,从而进行离线手动评分。另外,可以通过计算耗竭和基线测试之间的总差异来计算每个测量的高程得分。自动化的视频追踪设备可以用来替代视频评分。
    除了体积消耗之外,喷嘴式测厚仪还可用于评估舔微结构
  2. 对于具有来自每个主题的多个数据点(例如,,训练消耗数据或5分钟块数据)的分析,使用重复测量ANOVA。对于基于先验测试计划的来自每个主题的单个数据点的分析(例如,高程分数),可以使用广义线性模型。 Bonferroni校正后的广义线性模型跟踪了重要的相互作用。否则,所有分析都有一个拒绝标准: P < 0.05。
  3. 图3显示了在我们的盐食欲程序中(n = 19只雄性大鼠;组平均值±SEM;改编自Chang等人,2017)对于对照组大鼠的预期观察结果。在训练过程中,大鼠应该比盐饮用更多的蔗糖(图3A)。在耗竭钠后,与基线试验相比,耗竭试验期间大鼠应在盐配对情况下显示出花费的时间增加(图3B)。最后,在钠消耗后每只溶液与大鼠最后训练日相比,大鼠应该在盐摄入量和蔗糖消耗量方面显示出上升(图3C)。在耗竭和消耗测试过程中的神经操作可能揭示在钠耗竭后情境驱动的寻盐和盐消耗之间的分离。一个突出的例子是我们之前的研究(Chang等人,2017),在这个研究中,我们观察到在情境驱动的寻盐过程中存在特定的缺陷,但是在缺乏钠盐并伴随VP的光遗传抑制之后没有盐消耗。


    图3.我们盐分食欲程序的预期结果A.在训练过程中,大鼠应该比盐饮用更多的蔗糖。 B.在盐配对情况下,大鼠应该表现出消耗时间的增加以及在耗竭测试期间钠消耗后与蔗糖配对情境下花费的时间的减少。 C.与最后一个训练日相比,大鼠在消耗盐量和蔗糖消耗量方面有所上升,而钠消耗后每种溶液都有所增加。数据摘自Chang et al。,2017。

笔记

  1. 虽然我们使用球pt。当在每次会议开始之前将瓶子放置到位时,仍可能有一些泄漏的解决方案。为了最大限度地减少泄漏造成的溶液损失,我们在腔室底部的出口下方放置一个收集盘(,例如,一次性称重船),以收集在老鼠出现之前可能泄漏的任何溶液放入室内。一旦瓶子就位约5分钟,然后在大鼠被放入腔室之前将收集盘移除。然后将收集盘中的溶液添加到每次会话后留在瓶中的剩余物中,以提供对每次会话期间实际消耗的老鼠数量的更准确测量。
  2. 无论在基线测试期间与哪个解决方案配对,某些个体老鼠可能在每个环境中花费的时间方面表现出对该室的一个环境的偏见。然而,最重要的是在耗竭测试期间在每个环境中花费的时间的提升,这是衡量盐食欲现象的主要措施。我们一直观察到,在使用这种测量方法的对照大鼠中钠耗尽后,在盐配对环境中花费的时间强劲增加,尽管大鼠对一种情况或另一种情境可能具有任何固有偏差。
  3. 我们只使用雄性大鼠进行这一程序,但我们不希望看到雄性和雌性大鼠之间的差异。

食谱

  1. 对于培训课程,我们将每个解决方案制成1 L批次。因此,称取30g糖或盐并放入1L瓶中。然后用蒸馏水将瓶子充满1L。最后,加入2克无糖Kool-Aid粉(橙或葡萄)。然后混合溶液直至糖或盐(和Kool-Aid粉)溶解。
  2. 对于测试环节,1L批次只装满蒸馏水和2克Kool-Aid粉末。
  3. 呋塞米初始浓度为5%,用蒸馏水(例如5毫升呋塞米和20毫升蒸馏水)稀释至1%,浓度为10毫克/毫升。 br />

致谢

这项工作得到了美国国立卫生研究院拨款F32MH106178(SEC)和白厅基金研究基金2014-05-77(KSS)的资助。作者声明没有竞争的财务利益。该协议改编自Chang等人,2017年。

参考

  1. Chang,S.E.,Smedley,E.B.,Stansfield,K.J.,Stott,J.J。和Smith,K.S。(2017)。 腹侧苍白球神经元的光遗传学抑制损害情境驱动的寻盐。 J Neurosci 37(23):5670-5680。
  2. Galaverna,O. G.,Seeley,R.J.,Berridge,K.C.,Grill,H.J.,Epstein,A.N。和Schulkin,J。(1993)。 杏仁核核心的病变。 I:对味觉反应,味觉厌恶学习和钠食欲的影响。 Behav Brain Res 59(1-2):11-17。
  3. Hu,B.,Qiao,H.,Sun,B.,Jia,R.,Fan,Y.,Wang,N.,Lu,B。和Yan,J.Q.(2015)。 杏仁核中央核中的AT1受体阻断减弱了麝香醇对钠和水摄入的影响。 Neuroscience 307:302-310。
  4. Leshem,M.,Del Canho,S。和Schulkin,J。(1999)。 甲状旁腺切除大鼠的饥饿是特异性的。 Physiol Behav 67(4):555-559。
  5. Loriaux,A.L.,Roitman,J.D。和Roitman,M.F。(2011)。 Nucleus accumbens shell,但不是核心,跟踪盐的动机价值。 J Neurophysiol 106(3):1537-1544。
  6. Richter,C.P.(1936)。 肾上腺切除大鼠食盐量增加 Am J Physiol 115:155-161。
  7. Robinson,M.J。和Berridge,K.C。(2013)。 将学习的排斥立刻转化为激励“想要”。 Curr Biol 23(4):282-289。
  8. Roitman,M.F.,Na,E.,Anderson,G.,Jones,T.A。和Bernstein,I.L。(2002)。 诱导食欲改变伏隔核的树突形态并使大鼠对苯丙胺敏感。 J Neurosci 22(11):RC225。
  9. Schulkin,J。(2000)。 钙饥饿:行为和生物调节。 剑桥大学出版社。
  10. Seeley,R.J.,Galaverna,O.,Schulkin,J.,Epstein,A.N。和Grill,H.J。(1993)。 杏仁核核心的病变。 II:对口腔内NaCl摄取的影响。 行为脑解决方案59(1-2):19-25。
  11. &nbsp; Stouffer,E.M。和White,N.M。(2005)。 基于大鼠钠耗竭的潜在线索偏好 了解Mem 12(6):549-552。
  12. Tandon,S.,Simon,S.A。和Nicolelis,M.A。(2012)。 在盐剥夺期间的食欲改变与伏隔核,侧下丘脑和中央杏仁核中广泛的神经元适应平行。 Neurophysiol 108(4):1089-1105。
  13. Tindell,A.J.,Smith,K.S.,Berridge,K.C。和Aldridge,J.W。(2009)。 激励突出的动态计算:“想要”从未“喜欢”的东西。 J Neurosci 29(39):12220-12228。
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Copyright: © 2018 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. Chang, S. E. and Smith, K. S. (2018). Context-driven Salt Seeking Test (Rats). Bio-protocol 8(7): e2456. DOI: 10.21769/BioProtoc.2456.
  2. Chang, S. E., Smedley, E. B., Stansfield, K. J., Stott, J. J. and Smith, K. S. (2017). Optogenetic inhibition of ventral pallidum neurons impairs context-driven salt seeking. J Neurosci 37(23): 5670-5680.
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