Testing Effects of Chronic Chemogenetic Neuronal Stimulation on Energy Balance by Indirect Calorimetry

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The Journal of Neuroscience
May 2016



The fundamental of neuroscience is to connect the firing of neurons to physiological and behavioral outcomes. Chemogenetics enables researchers to control the activity of a genetically defined population of neurons in vivo through the expression of designer receptor exclusively activated by designer drug (DREADD) in specific neurons and the administration of its synthetic ligand clozapine N-oxide (CNO) (Sternson and Roth, 2014). Using stimulatory Gq-coupled DREADD (hM3Dq) in mice, we showed that leptin receptor (LepRb)-expressing neurons in the preoptic area (POA) of the hypothalamus are warm-sensitive neurons that mediate warm-responsive metabolic and behavioral adaptations by reducing energy expenditure and food intake (Yu et al., 2016). We also used DREADD technology to test effects of chronic stimulation of POA LepRb neurons on energy expenditure, food intake, and body weight with the TSE indirect calorimetry system. Here we describe the detailed protocol of how we used indirect calorimetry to study the outcome of chronic stimulation of POA LepRb neurons. This protocol can be adapted to study long-term metabolic and behavioral consequences of other neuronal modulations, with possible modifications to the type of DREADD, duration of CNO treatment, or method of CNO delivery.

Keywords: Chemogenetics (化学遗传学), DREADD (DREADD), Energy expenditure (能量消耗), Food intake (食物摄入量), Indirect calorimetry (间接量热法), TSE (TSE)


The POA is a central hub for body temperature homeostasis, which receives thermosensory information from the periphery and tunes the degree of sympathetic output to brown adipose tissue (BAT), cutaneous blood vessels, and heart to control the amount of heat generation and dissipation (Nakamura, 2011). We discovered that LepRb neurons in the POA are stimulated by warm ambient temperature and mediate warm-adaptive responses that include suppression of BAT thermogenesis and food intake (Yu et al., 2016). This discovery was made mainly through chemogenetic stimulation of POA LepRb neurons by virally expressing Gq-coupled DREADD, hM3Dq, in POA LepRb neurons and injecting CNO in mice. A single IP injection of CNO can stimulate target neurons up to 10 h (Krashes et al., 2011; Rezai-Zadeh et al., 2014). This long-lasting CNO effect allows researchers to modulate target neuron activity chronically with two IP injections of CNO per day.

Optogenetics and chemogenetics have revolutionized the field of neuroscience by providing tools to selectively manipulate target neuron activity with its own unique advantages and disadvantages. For studying neurons that modulate energy balance, researchers often use an indirect calorimetry system to simultaneously measure energy expenditure and food intake for an extended period of time (Rezai-Zadeh et al., 2014; Correa et al., 2015; Qualls-Creekmore et al., 2017). Chemogenetics is best suited for this type of study thanks to slow clearance of CNO from the body and no requirement of optical devices as in optogenetics. In our study, we investigated consequences of chronic stimulation of POA LepRb neurons by DREADD with the PhenoMaster indirect calorimetry system. CNO was injected at 0.3 mg/kg twice per day for six days, and energy expenditure and food intake were continuously measured every 25 min. Body weight was measured once every morning to monitor how changed energy expenditure and food intake affected body weight. In this protocol, we describe a step-by-step procedure of using the TSE PhenoMaster indirect calorimetry system to measure energy expenditure and food intake during chronic stimulation of POA LepRb neurons by DREADD in mice.

Materials and Reagents

  1. ½ CC Lo-dose U-100 insulin syringe 28 G ½ (BD, catalog number: 329461 )
  2. 1.5 ml microcentrifuge tube (SARSTEDT, catalog number: 72.690.301 )
  3. LepRb-Cre mice
    1. In-house bred and derived from original breeders kindly provided by Dr. Martin Myers, Jr., University of Michigan (Leshan et al., 2006); that were injected with AAV5-hSyn-DIO-mCherry (Vector Core, University of North Carolina at Chapel Hill) in the POA (control group, 3 month old, 3 males and 2 females, n = 5).
    2. LepRb-Cre mice that were injected with AAV5-hSyn-DIO-hM3Dq-mCherry (Vector Core, University of North Carolina at Chapel Hill, kindly made available by Dr. Bryan Roth) in the POA (experimental group, 3 month old, 3 males and 2 females, n = 5).
    3. All experiments were approved by the Institutional Animal Care and Use Committee at Pennington Biomedical Research Center.
  4. CNO (Sigma-Aldrich, catalog number: C0832 )
  5. Saline/0.9% NaCl (B. Braun Medical, catalog number: L8001 )
  6. CNO stock (see Recipes)
  7. CNO working solution (see Recipes)


  1. PhenoMaster (TSE systems)
  2. Scale (Ohaus, Scout-Pro, model: SPE601 )
  3. Vortex Mixer (American Scientific Products, catalog number: S8223-1 )


  1. LabMaster V5.0.8 (TSE systems)
  2. Excel 2010 (Microsoft)
  3. SPSS 22 (IBM)


  1. Setting up the PhenoMaster TSE system
    1. Measure body weight of each mouse that will be used for the experiment.
      Note: Age of mice was matched to eliminate the confounding effect of age on energy expenditure and food intake and to minimize the body weight difference between mice. At the beginning of the experiment, mouse body weight ranged between 20 g and 30 g. It is important to use mice with similar body composition to minimize errors during data analysis when body weight is used to normalize energy expenditure and food intake.
    2. Open LabMaster and calibrate gas sensors for O2 and CO2, and weight scales for food and water according to manufacturer’s manual. It is not necessary to turn on the system before calibration.
    3. Turn on climate chambers and set the temperature at 23 °C (Figure 1).

      Figure 1. The PhenoMaster TSE system. One climate chamber can harbor 6 boxes that can measure O2, CO2, food intake, water consumption.

    4. Transfer mice to housing boxes (1 mouse/box; Figure 2) and securely seal lids using 2 latches per cage (one front and one back; Figure 2).

      Figure 2. A mouse housing box for the PhenoMaster TSE system. Each box is equipped with tubing for gas in and out, weight sensors for water and food, and an infrared light beam frame for locomotor activity.

    5. Fill water bottles with tap water and food hoppers with regular chow, and hang them at corresponding weight sensors. It is important to check each water bottle and food hopper for good water flow and accessibility of food pellets, respectively.
    6. Under the ‘Setup’ tab in LabMaster, input animal IDs, body weight, and set up other parameters such as a measurement interval. In our experiment, the measurement interval was 25 min.
    7. Under the ‘Measurement’ tab, select ‘Start’ to begin measurements.

  2. Daily injections and body weight measurement
    1. The room had a 12:12 light:dark cycle with the light on at 7 AM and off at 7 PM.
    2. Whenever opening a box to measure body weight, refill water or food, or perform injections, it is important not to open the lid while the system is measuring that specific box to prevent outside air from coming into the box. It is safest to open the lid of a box right after the system has just finished measuring that box. Select ‘Status > Calo’ in LabMaster to identify the box the system is actively measuring.
    3. Before opening the box, select ‘Status > Trial Monitor Drink/Feed’ in LabMaster to open the box monitoring screen (Figure 3). Right click on the green circle underneath a box number, and then select ‘Pause Box’ to temporarily pause measurements of that box (Figure 3). The circle turns yellow. Once the box lid is back on and sealed, right click the same circle and select ‘Continue Box’ to resume measurements (Figure 4).
    4. Once a week during the experiment, replace dirty mouse box bottoms with clean ones and refill water bottles with fresh water.
    5. When an accident happens, such as opening of a box while the system is reading the same box, data from that time point for that box should not be used for later analysis.

    Figure 3. Example image of Trial Monitor Drink/Feed screen. The green circles under box numbers indicate boxes included for measurements. In this example, boxes #12 and #24 are not included in the experiment.

    Figure 4. Example image showing how to continue measurements of a temporarily paused box

    1. For overall experimental scheme, see Figure 5.

      Figure 5. Overall experimental scheme. Mice were first acclimated to a new environment and then injected with 3 days of saline followed by 6 days of CNO. Body weight was measured every morning.

    2. Day 1-3: Let mice acclimate to a new environment without any disturbance other than refilling water or food. During this period, if water leaks and floods the bedding, replace the faulty parts (e.g., sipper or entire bottle) and the box bottom.
    3. Day 4-15: body weight measured daily at 9 AM for each mouse.
    4. Day 4-6: saline IP injection twice daily at 9:30 AM and 6 PM for each mouse. Detemin the amount of saline injection by the body weight of the day, such that it matches the amount of CNO working solution (0.06 mg/ml) that would have been needed for that body weight to achieve the dose of 0.3 mg/kg. For example, 20 g mouse receives 100 μl of saline.
    5. Day 7-12: CNO IP injection (0.3 mg/kg) twice daily at 9:30 AM and 6 PM for each mouse. The amount of CNO injection is determined by the body weight of the day as described above.
    6. Day 13-15: mice recover from daily injections (no injection).

  3. Finishing the experiment and retrieving data
    1. On day 14 after morning body weight measurement, stop measurements by selecting ‘Measurement > Stop’ in LabMaster.
    2. Take out mice and put them in regular housing cages with water and food.
    3. To export data, first go to ‘View’ to select parameters and box numbers to export data.
    4. Select ‘Export > Table’, then designate file name and location.

Data analysis

  1. Open the exported file in Microsoft Excel (Figure 6A).
  2. Copy energy expenditure (H(1) in kcal/h/kg) and food intake (Feed in g) data to a new tab or file.
  3. Make a table in Excel for daily body weight (Figure 6B).

    Figure 6. Example tables for data analysis. A. A part of the energy expenditure data table that was exported by LabMaster. B. The body weight table that shows daily body weight of all mice, group means, and standard errors of the means during the experiment.

  4. Divide data based on the group and calculate means and standard errors for all time points in each group.
  5. For body weight analysis, calculate body weight change for each day in reference to the body weight on the first CNO injection day.
  6. For energy expenditure and food intake analysis, calculate daily means (kcal/g/day) separately for saline injection (3 days), CNO injection (6 days), and recovery (2 days) (Figure 7).
  7. Perform statistical analysis by repeated measures ANOVA (analysis of variance) in SPSS. Multiple pairwise comparisons were corrected by the Bonferroni method.

    Figure 7. The table for daily food intake analysis. Daily food intake was averaged for each treatment condition, and group means were compared.


Homozygous LepRb-Cre mice were used to maximize Cre expression for more efficient viral gene expression.


  1. CNO stock (5 mg/ml)
    1. Add 1 ml saline to 5 mg of CNO powder
    2. Vortex until the powder is fully dissolved
    3. Transfer to a 1.5 ml microcentrifuge tube and keep the stock at 4 °C
    Note: Stock CNO is stable at 4 °C for 6 months at least. However, CNO may precipitate over time. Adding a small amount of DMSO (3-10%) may help dissolving CNO and preventing precipitation.
  2. CNO working solution (0.06 mg/ml)
    1. Take out and equilibrate the CNO stock solution to room temperature
    2. Vortex to mix and dissolve any possible CNO pellets
    3. Dilute the CNO stock 83.3-fold with saline to achieve a final concentration of 0.06 mg/ml. For example, to make 1 ml working solution, add 83 μl CNO stock to 917 μl saline
    4. Make it fresh on the day of injection and use it as room temperature solution without warming it up to body temperature 


This work was supported by AHA053298N, P/F DK020572-30, R01DK092587 (HM), P20GM103528 (HM and SY), and 2P30-DK072476 (HM and SY). Partial support was provided through the Animal Phenotyping Core funded by NIDDK NORC Center Grant P30 DK072476. The authors declare no competing financial interests. This protocol is adapted from procedures published in Yu et al. (2016).


  1. Correa, S. M., Newstrom, D. W., Warne, J. P., Flandin, P., Cheung, C. C., Lin-Moore, A. T., Pierce, A. A., Xu, A. W., Rubenstein, J. L. and Ingraham, H. A. (2015). An estrogen-responsive module in the ventromedial hypothalamus selectively drives sex-specific activity in females. Cell Rep 10: 62-74.
  2. Krashes, M. J., Koda, S., Ye, C., Rogan, S. C., Adams, A. C., Cusher, D. S., Maratos-Flier, E., Roth, B. L. and Lowell, B. B. (2011). Rapid, reversible activation of AgRP neurons drives feeding behavior in mice. J Clin Invest 121(4): 1424-1428.
  3. Leshan, R. L., Bjornholm, M., Munzberg, H. and Myers, M. G., Jr. (2006). Leptin receptor signaling and action in the central nervous system. Obesity (Silver Spring) 14 Suppl 5: 208S-212S.
  4. Nakamura, K. (2011). Central circuitries for body temperature regulation and fever. Am J Physiol Regul Integr Comp Physiol 301(5): R1207-1228.
  5. Qualls-Creekmore, E., Yu, S., Francois, M., Hoang, J., Huesing, C., Bruce-Keller, A., Burk, D., Berthoud, H. R., Morrison, C. D. and Munzberg, H. (2017). Galanin-expressing GABA neurons in the lateral hypothalamus modulate food reward and noncompulsive locomotion. J Neurosci 37: 6053-6065.
  6. Rezai-Zadeh, K., Yu, S., Jiang, Y., Laque, A., Schwartzenburg, C., Morrison, C. D., Derbenev, A. V., Zsombok, A. and Munzberg, H. (2014). Leptin receptor neurons in the dorsomedial hypothalamus are key regulators of energy expenditure and body weight, but not food intake. Mol Metab 3(7): 681-693.
  7. Sternson, S. M. and Roth, B. L. (2014). Chemogenetic tools to interrogate brain functions. Annu Rev Neurosci 37: 387-407.
  8. Yu, S., Qualls-Creekmore, E., Rezai-Zadeh, K., Jiang, Y., Berthoud, H. R., Morrison, C. D., Derbenev, A. V., Zsombok, A. and Munzberg, H. (2016). Glutamatergic preoptic area neurons that express leptin receptors drive temperature-dependent body weight homeostasis. J Neurosci 36(18): 5034-5046.


神经科学的基础是将神经元的激发与生理和行为结果联系起来。化学遗传学使研究人员能够通过在特定神经元中由设计药物(DREADD)专门激活的设计受体的表达以及其合成配体氯氮平N-乙酰半胱氨酸的施用来控制基因定义的体内神经元群体的活性氧化物(CNO)(Sternson和Roth,2014)。在小鼠中使用刺激性Gq偶联的DREADD(hM3Dq),我们发现在下丘脑的视前区(POA)中表达瘦素受体(LepRb)的神经元是通过降低能量介导热反应性代谢和行为适应性的热敏感神经元支出和食物摄入量(Yu等人,2016年)。我们还使用DREADD技术来测试POE LepRb神经元对TSE间接量热系统的能量消耗,食物摄入量和体重的慢性刺激作用。在这里我们描述了我们如何使用间接量热法研究POA LepRb神经元的慢性刺激结果的详细方案。该协议可适用于研究其他神经元调节的长期代谢和行为后果,并可能对DREADD类型,CNO治疗持续时间或CNO递送方法进行修改。

【背景】POA是体温平衡的中心枢纽,它从外围接收热敏感信息并调节交感输出到棕色脂肪组织(BAT),皮肤血管和心脏的程度以控制热量产生和消散的量(Nakamura ,2011)。我们发现POA中的LepRb神经元受到温暖的环境温度的刺激并介导包括抑制BAT产热和食物摄取的温暖适应性反应(Yu等人,2016)。这一发现主要是通过在POA LepRb神经元中病毒表达Gq偶联的DREADD,hM3Dq和在小鼠中注射CNO,通过POA LepRb神经元的化学基因刺激来实现的。单次IP注射CNO可刺激靶神经元达10小时(Krashes等人,2011; Rezai-Zadeh等人,2014)。这种长期持续的CNO作用使研究人员能够通过每天两次IP注射CNO来长期调节靶神经元活动。

光遗传学和化学遗传学已经通过提供工具来选择性地操纵目标神经元活动,并具有其独特的优点和缺点,从而革新了神经科学领域。为了研究调节能量平衡的神经元,研究人员经常使用间接量热系统来同时测量能量消耗和食物摄入量(Rezai-Zadeh et al。,2014; Correa et al。,2015; Qualls-Creekmore 等,2017)。化学遗传学最适合这种类型的研究,这要归功于CNO从体内缓慢清除,并且不需要像光遗传学那样的光学装置。在我们的研究中,我们用PhenoMaster间接测热系统研究了DREADD对POA LepRb神经元的慢性刺激的后果。 CNO每天注射0.3 mg / kg,持续6天,每25分钟持续测量能量消耗和食物摄入量。每天早晨测量一次体重,以监测能量消耗和食物摄入量如何改变对体重的影响。在这个协议中,我们描述了使用TSE PhenoMaster间接量热系统来测量小鼠中DREADD慢性刺激POA LepRb神经元期间的能量消耗和食物摄入量的逐步程序。

关键字:化学遗传学, DREADD, 能量消耗, 食物摄入量, 间接量热法, TSE


  1. ½CC低剂量U-100胰岛素注射器28 G½(BD,目录号:329461)

  2. 1.5 ml微量离心管(SARSTEDT,目录号:72.690.301)
  3. LepRb-Cre 小鼠
    1. 由密歇根大学Martin Myers,Jr.博士友好提供的原始育种家培育的内部品种(乐山等,2006);在POA(对照组,3个月大,3名男性和2名女性,n = 5)中注射AAV5-hSyn-DIO-mCherry(载体核心,北卡罗来纳大学教堂山分校)。 br />
    2. 在POA(试验组,3个月)内注射AAV5-hSyn-DIO-hM3Dq-mCherry(Vector Core,University of North Carolina at Chapel Hill,由Bryan Roth博士友情提供)的LepRb- 3岁男性,2女性,n = 5)。
    3. 所有实验均得到了Pennington生物医学研究中心的动物管理和使用委员会的批准。
  4. CNO(Sigma-Aldrich,目录号:C0832)
  5. 盐水/ 0.9%NaCl(B.Braun Medical,目录号:L8001)
  6. CNO股票(见食谱)
  7. CNO工作解决方案(见食谱)


  1. PhenoMaster(TSE系统)
  2. 量表(Ohaus,Scout-Pro,型号:SPE601)
  3. 涡旋混合器(美国科学产品,目录号:S8223-1)


  1. LabMaster V5.0.8(TSE系统)
  2. Excel 2010(微软)
  3. SPSS 22(IBM)


  1. 设置PhenoMaster TSE系统
    1. 测量将用于实验的每只小鼠的体重。
    2. 根据制造商的手册打开LabMaster并校准O 2和CO 2的气体传感器和食品和水的重量标尺。
    3. 打开气候室并将温度设置在23°C(图1)。

      图1. PhenoMaster TSE系统一个气候箱可以包含6个盒子,可以测量O 2,CO 2,食物摄入量,耗水量。

    4. 将小鼠转移到住房箱(1只鼠标/箱;图2),并使用每个笼2个闩锁(一个前部和一个后部;图2)牢固地密封盖子。

      图2. PhenoMaster TSE系统的鼠标收纳盒每个盒子都配有进出气管,水和食物重量传感器,以及用于运动活动的红外光束框。 br />
    5. 用自来水和普通食物的料斗填充水瓶,并将其悬挂在相应的重量传感器上。检查每个水瓶和食物料斗以分别获得良好的水流量和食物颗粒的可接近性是很重要的。
    6. 在LabMaster的'设置'选项卡下,输入动物ID,体重,并设置其他参数,如测量间隔。在我们的实验中,测量间隔为25分钟。
    7. 在“测量”标签下,选择“开始”开始测量。

  2. 每日注射和体重测量
    1. 房间有一个12:12的光:黑暗循环,早上7点亮,晚上7点关闭。
    2. 每当打开一个盒子来测量体重,补充水或食物或进行注射时,在系统测量特定盒子时防止外部空气进入盒子是非常重要的。系统刚刚完成对盒子的测量后,打开盒子的盖子是最安全的。选择'状态> LabMaster中的Calo'来识别系统正在测量的盒子。
    3. 在打开框之前,请选择'状态>在LabMaster中试用监控饮料/喂料“来打开箱子监控屏幕(图3)。右键单击盒号下方的绿色圆圈,然后选择“暂停盒”暂时暂停该盒的测量(图3)。圆圈变成黄色。一旦箱盖重新打开并密封,右击同一圆圈并选择“继续箱子”重新开始测量(图4)。
    4. 实验过程中每隔一周进行一次,将脏兮兮的鼠标盒底部更换为干净的底部,并用淡水重新装满水瓶。
    5. 发生事故时,例如在系统正在读取同一个盒子时打开盒子,该盒子的该时间点的数据不应该用于以后的分析。

    图3. Trial Monitor Drink / Feed屏幕的示例图像框编号下方的绿色圆圈表示用于测量的框。在这个例子中,框#12和#24不包含在实验中。


    1. 整体实验方案请参见图5.


    2. 第1-3天:让小鼠适应新环境,除了补充水分或食物外,没有任何干扰。在此期间,如果水渗漏并淹没床上用品,请更换有故障的部件(例如,吸管或整个瓶子)和箱子底部。
    3. 第4-15天:每天早上9点测量每只小鼠的体重。
    4. 第4-6天:每天两次在上午9:30和下午6:00对每只小鼠进行盐水IP注射。根据当天体重测定生理盐水注射量,以使其与体重达到0.3mg / kg剂量所需的CNO工作溶液量(0.06mg / ml)相匹配。例如,20克小鼠接受100μl盐水。
    5. 第7-12天:CNO IP注射(0.3mg / kg),每天两次,上午9:30和下午6点,每只小鼠。如上所述,CNO注射量由当天的体重决定。
    6. 第13-15天:小鼠从每日注射中恢复(不注射)。

  3. 完成实验并检索数据
    1. 在早晨体重测量后的第14天,通过选择'测量>停止“在LabMaster中。
    2. 把老鼠拿出来,用水和食物把它们放在正规的笼子里。
    3. 要导出数据,首先进入'查看'选择参数和框号以导出数据。
    4. 选择“导出>表',然后指定文件名和位置。


  1. 在Microsoft Excel中打开导出的文件(图6A)。
  2. 将能量消耗(H(1)以千卡/小时/千克为单位)和食物摄入量(以千克为单位)数据复制到新标签或文件中。

  3. 在Excel中为每日体重制作一张桌子(图6B)。

    图6.用于数据分析的示例表 A.由LabMaster导出的能源消耗数据表的一部分。 B.体重表显示了所有小鼠的每日体重,群体平均值和实验过程中标准误差。

  4. 根据组划分数据并计算每组中所有时间点的均值和标准误差。
  5. 对于体重分析,根据第一个CNO注射日的体重计算每一天的体重变化。
  6. 对于能量消耗和食物摄入量分析,分别计算盐水注射(3天),CNO注射(6天)和恢复(2天)(图7)的每日平均值(千卡/克/天)。
  7. 通过SPSS中的重复测量ANOVA(方差分析)进行统计分析。通过Bonferroni方法校正了多对配对比较。





  1. CNO库存(5毫克/毫升)

    1. 加入1毫升生理盐水至5毫克CNO粉末
    2. 漩涡直到粉末完全溶解
    3. 转移到1.5毫升微量离心管中,并保持在4°C
  2. CNO工作溶液(0.06mg / ml)
    1. 取出并平衡CNO储备溶液至室温
    2. 涡旋混合并溶解任何可能的CNO颗粒
    3. 用盐水稀释CNO储液83.3倍以达到0.06mg / ml的终浓度。例如,要制造1 ml工作溶液,将83μlCNO储液加入917μl生理盐水中
    4. 在注射当天使其保持新鲜状态,并将其作为室温溶液使用,而不会升温至体温范围内


这项工作得到AHA053298N,P / F DK020572-30,R01DK092587(HM),P20GM103528(HM和SY)和2P30-DK072476(HM和SY)的支持。部分支持通过由NIDDK NORC中心Grant P30 DK072476资助的动物表型鉴定核心提供。作者声明没有竞争的财务利益。该协议是根据Yu等人发表的程序改编的。


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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. Yu, S. and Munzberg, H. (2018). Testing Effects of Chronic Chemogenetic Neuronal Stimulation on Energy Balance by Indirect Calorimetry. Bio-protocol 8(8): e2811. DOI: 10.21769/BioProtoc.2811.
  2. Yu, S., Qualls-Creekmore, E., Rezai-Zadeh, K., Jiang, Y., Berthoud, H. R., Morrison, C. D., Derbenev, A. V., Zsombok, A. and Munzberg, H. (2016). Glutamatergic preoptic area neurons that express leptin receptors drive temperature-dependent body weight homeostasis. J Neurosci 36(18): 5034-5046.