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Jul 2020

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Generation of Mouse Primary Hypothalamic Neuronal Cultures for Circadian Bioluminescence Assays
培养小鼠原发性下丘脑神经元用于昼夜生物发光分析   

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Abstract

An endogenous circadian clock system enables organisms to adapt to time-of-day dependent environmental changes. In consequence, most physiological processes exhibit daily rhythms of, e.g., energy metabolism, immune function, sleep, or hormone production. Hypothalamic circadian clocks have been identified to play a particular role in coordinating many of these processes. Primary neuronal cultures are widely used as a physiologically relevant model to study molecular events within neurons. However, as circadian rhythms include dynamic molecular changes over longer timescales that vary between individual cells, longitudinal measurement methods are essential to investigate the regulation of circadian clocks of hypothalamic neurons. Here we provide a protocol for generating primary hypothalamic neuronal cultures expressing a circadian luciferase reporter. Such reporter cells can be used to longitudinally monitor cellular circadian rhythms at high temporal resolution by performing bioluminescence measurements.

Keywords: Primary hypothalamic neurons (原发性下丘脑神经元), Circadian clocks (昼夜节律生物钟), Luciferase reporter (萤光素酶报告基因), Bmal1 (Bmal1), Circadian rhythms (昼夜节律)

Background

To adapt to recurring time-of-day dependent changes in their environment, many organisms have developed an endogenous circadian clock system that regulates 24-h rhythms of behavioral and physiological processes (Sharma, 2003). In mammals, a master circadian pacemaker resides in the hypothalamic suprachiasmatic nucleus (SCN). It coordinates cellular clock regulation throughout the body with external time. Daily patterns of sleep, appetite, and metabolism are regulated by cellular circadian clocks residing in hypothalamic neurons (Cedernaes et al., 2019).


In mammalian cells, circadian clocks consist of interlocked transcriptional-translational feedback loops (TTFLs). In the core TTFL, the transcription factors circadian locomotor output cycles kaput (CLOCK) and brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (BMAL1 or ARNTL) activate the expression of their own repressors, period (PER1-3) and cryptochrome (CRY1/2), which leads to circadian oscillations of gene expression and abundance of protein products (Ko and Takahashi, 2006).


Methods to directly quantify mRNA and protein levels, such as quantitative PCR or Western blotting, require repetitive sampling from different individuals or preparations at different time points, which is labor-intensive, and, in case of short time intervals or low amounts of cellular material, highly impractical (Yu and Hardin, 2007). To overcome these problems, circadian reporters, where the expression of the firefly luciferase enzyme is under the control of a clock gene promoter (e.g., Bmal1 or Per2), have been developed (Brown et al., 2005; Ramanathan et al., 2012; Fang et al., 2017). They allow real-time tracking of cellular circadian rhythms by performing bioluminescence measurements and therefore are widely used to study circadian clock function (Ramanathan et al., 2012).


Immortalized cell lines are valuable tools for molecular biological research, as they are readily available and can be expanded without limitations (Pan et al., 2009). In recent years, they have become established in-vitro models for the investigation of hypothalamic clocks (Fick et al., 2010 and 2011; Tsang et al., 2020). However, since immortalized cell lines are genetically and phenotypically different from their tissue origins, data obtained from such systems has to be interpreted critically and a verification with a more physiologically relevant model system is advised (Pan et al., 2009).


Primary neuronal cultures are obtained directly from the animal’s central nervous system and maintain many physiological and biochemical characteristics of their tissue origin (Gordon et al., 2013; Verma et al., 2020). Therefore, they are useful model systems to address physiological relevance. Here we provide a protocol for the generation of primary hypothalamic neuron cultures stably transduced with a circadian Bmal1-luciferase reporter. Creation of these reporter cells allows observation and quantitative description of hypothalamic neuronal circadian rhythms in real-time, avoiding labor-intensive and material-consuming biochemical experiments. These cells can further be used to investigate clock resetting effects of various factors such as hormones or metabolites.


Materials and Reagents

  1. 96-well plate (Corning, catalog number: 3610)

  2. BD Falcon cell strainer, 70 µm (BD Biosciences, catalog number: 352350)

  3. 100-mm culture dish

  4. 35-mm dish

  5. 15-ml Falcon tube

  6. 10-ml serological pipette

  7. Fire-polished glass pipette

  8. 2-8-month-old male and female C57BL/6J mice

  9. Bmal1-luciferase lentivirus (Brown et al., 2005)

  10. Poly-D-lysine hydrobromide (Millipore Sigma, catalog number: P6407)

  11. Laminin (BD Biosciences, catalog number: 354232)

  12. Hank’s balanced salt solution, HBSS (PAA, catalog number: H15-008)

  13. Earle’s balanced salt solution, EBSS, with Phenol Red (Thermo Fisher Scientific, Gibco, catalog number: 24010043)

  14. Papain, suspension (Worthington Biochemical Corporation, catalog number: LS003126)

  15. Deoxyribonuclease I, DNaseI (Worthington Biochemical Corporation, catalog number: LS002058)

  16. Neurobasal medium, minus phenol red (Thermo Fisher Scientific, Gibco, catalog number: 12348017)

  17. B-27 supplement (Thermo Fisher Scientific, Gibco, catalog number: 17504044)

  18. GlutaMAX supplement (Thermo Fisher Scientific, Gibco, catalog number: 35050061)

  19. Ovomocoid (Worthington Biochemical Corporation, catalog number: LS003085)

  20. Bovine serum albumin (MilliporeSigma, catalog number: A7030)

  21. L-cysteine (MilliporeSigma, catalog number: C7352)

  22. Fetal bovine serum, FBS (Thermo Fisher Scientific, Gibco, catalog number: 10500-064)

  23. Trypan blue solution (MilliporeSigma, catalog number: T8154)

  24. Cytosine β-D-arabinofuranoside, AraC (Millipore Sigma, catalog number: C1768)

  25. Pasteur pipettes (Th. Geyer, catalog number: 7691061)

  26. 2-Mercaptoethanol (Millipore Sigma catalog number: M3148)

  27. Adhesive clear PCR seal (Biozym, catalog number: 600208)

  28. D-luciferin sodium salt (Applichem, catalog number: A1006)

  29. Polybrene, Hexadimethrine bromide (Millipore Sigma, catalog number: H9268)

Equipment

  1. Sterile laminar flow hood (Thermo Fisher Scientific, model: MSC-Advantage, catalog number: 51025411)

  2. Multimode microplate reader (Berthold Technologies, model: Tristar LB941)

  3. Fluorescence microscope (Nikon, model: Eclipse Ts2R)

  4. LED unit (Nikon, model: C-LEDFL470)

  5. Fluorescence filter cube (Nikon, model: C-LED470, Excitation: 470/40 nm, Dichroic: 500 nm, Emission: 535/55 nm)

  6. 10× objective (Nikon, model: CFI Achromat ADL-10×, NA: 0.4)

  7. 20× objective (Nikon, model: CFI Achromat LWD ADL-20×, NA: 0.4)

  8. Hemocytometer (Laboroptik, model: Neubauer)

  9. Water bath (GFL, model: 1002)

  10. Dissecting microscope (Leica, model: MZ6)

  11. Dissection scissors (Fine Science Tools, catalog number: 91402-14)

  12. Dissection forceps (Fine Science Tools, catalog number: 11000-12)

  13. No.11 scalpel (Feather, catalog number: 200210011)

  14. No.5 sharp forceps (Fine Science Tools, catalog number: 11252-20)

  15. 2 × curved forceps (Fine Science Tools, catalog number: 11274-20)

  16. Student Fine Scissors (Fine Science Tools, catalog number: 91460-11)

Software

  1. MicroWin 2000 (Labsis, https://labsis.de)

Procedure

  1. Schedule matings

    1. To generate E15-16 embryos, schedule the mating day of the adult mice 15-16 d before dissection. We use 2-8-month-old adult C57BL/6J mice for mating.

    2. On the next morning, confirm successful mating by vaginal plug check as described previously (Behringer et al., 2016).

    3. Confirm pregnancy by palpitation or visually before dissection.


  2. Reagent preparation

    Note: Avoid repeated freeze-thaw cycles for labile reagents.

    1. Prepare poly-D-lysine (PDL) stock solution (500 µg/ml). Divide into 5-ml aliquots and store at -20 °C.

    2. Prepare laminin stock solution (0.1 µg/µl) in PBS. Divide into 250-µl aliquots and store at -20 °C.

    3. Prepare ovo/albumin inhibition solution, containing 6 mg ovomucoid and 6 mg bovine serum albumin in 6 ml EBSS. Store at 4 °C until use.

    4. Prepare DNaseI solution, with 1,000 U DNase I in 500 µl EBSS and store at -20 °C.

    5. Prepare plating medium (Neurobasal + 2% (v/v) B27 + 2 mM Glutamax + 10% (v/v) FBS + 1× penicillin/streptomycin) and store at 4 °C until use. Equilibrate at 37 °C before adding it to the culture.

    6. Prepare feeding medium (same as plating medium, but without FBS) and store at 4 °C until use. Equilibrate at 37 °C before adding it to the culture.

    7. Prepare digestion solution (100 U Papain, 5 ml EBSS, 1.1 mM EDTA (11 µl 0.5 M EDTA), 5.5 mM cysteine (3.3 mg), 0.067 mM 2-Mercaptoethanol (2.34 µl 1 % (v/v)/14.3 M pure liquid) and 250 µl DNaseI solution), sterile filtrate with a 0.2-µm filter. Activate at 37 °C for 20-30 min before use.

    8. Prepare resuspension medium (2.7 ml EBSS, 300 µl ovo/albumin inhibition solution, 150 µl DNaseI solution).


  3. PDL and laminin double coating

    1. One day before dissection coat 96-well plates with PDL and laminin.

    2. Dilute PDL stock solution with PBS to working concentration 50 µg/ml. Mix 0.5 ml PDL stock solution (500 µg/ml) with 4.5 ml PBS to prepare 5 ml diluted PDL solution (50 µg/ml).

    3. Filter with 0.2-µm filter before use.

    4. Coat the culture surface of a 96-well plate with 7.5 µg/cm2 PDL. Cover the wells of a 96-well plate with 48 µl diluted PDL solution (50 µg/ml).

    5. Incubate overnight at room temperature (or at least for 2 h at 37 °C).

    6. Wash 3 times with 100 µl sterile H2O.

    7. Allow to dry completely under a sterile cell culture hood.

    8. Thaw laminin stock solution slowly at 2-8 °C.

    9. Dilute laminin stock solution with PBS to a working concentration of 6.4 µg/ml. Mix 320 µl laminin stock solution (0.1 µg/µl) with 4.68 ml PBS to prepare 5 ml diluted laminin solution (6.4 µg/ml).

    10. Cover the PDL-coated wells with 50 µl diluted laminin solution (6.4 µg/ml). The final coating concentration will be around 1 µg/cm2.

    11. Incubate for 2 h at 37 °C.

    12. Wash 3 times with sterile 100 µl PBS, add 100 µl plating medium and store at 37 °C and 5 % CO2 in the incubator.


  4. Dissection

    Note: A swiftly executed preparation procedure is crucial for cell viability. Consider practicing the procedure several times before preparing experimental samples. Before starting the dissection, ensure that required materials are in place and that all equipment is disinfected.

    1. Use embryonic day 15 to 16 (E15-16) old embryos from a pregnant mother (C57BL/6J mothers usually have 8-10 pups).

    2. Work under a laminar flow hood and always apply aseptic techniques to reduce the risk of contamination with bacteria, fungi, and mycoplasma.

    3. Sacrifice mother by cervical dislocation, open the abdominal cavity with dissection scissors and forceps. The embryos are located at the posterior part of the abdominal cavity.

    4. Carefully remove the uterus horns with two curved forceps with gentle opposite pulling motions and transfer them into a 100-mm culture dish filled with ice-cold HBSS.

    5. Extract embryos with dissection scissors and curved forceps and transfer them into a new 100-mm culture dish with ice-cold HBSS.

    6. Decapitate the embryos with dissection scissors.

    7. Hold the head in position by piercing No. 5 sharp forceps into orbital cavities and by pushing the rostral part of the brain down. Simultaneously use curved forceps to remove the outer skin and skull, by peeling them gently off from caudal to rostral. Start with the left hemisphere and then repeat that step with the right hemisphere. Carefully remove the brain with curved forceps and deposit it into a 35-mm Petri dish with ice-cold HBSS. Repeat Steps D5 to D7 for the other embryos.

      Note: Avoid applying pressure onto the tissue while extracting the brains, to maintain brain integrity. Furthermore, take care that the brains are always covered with medium, do not let them dry out.

    8. Under a dissecting microscope, isolate the hypothalami with forceps and scalpel (see Figure 1 for details). Collect them in a 35-mm dish with ice-cold HBSS and keep on ice. Use a cut 1,000-µl pipette tip or a dropper with a wide opening to transfer the dissected tissue.



      Figure 1. Steps for hypothalamus dissection from intact E16 brains. A. Turn the brain over so that the ventral part is facing upwards. Remove the caudal part of the brain by making a coronal cut at the posterior border of the mammillary bodies. Make a second coronal cut ~1.5 mm anterior from the first to remove the rostral part of the brain. B. Rotate the remaining brain to get a coronal orientation and make sure that the rostral part is facing upwards. Dissect the hypothalamic tissue block: make two lateral cuts ~0.5 mm each from the midline and one additional cut ventral to the anterior commissure (ac). Red dashed lines indicate the cutting positions. The dissected area is highlighted in blue. Further abbreviations: LV – lateral ventricle, Thal – thalamus, Hypo – hypothalamus, Teg – tegmentum, Med – medulla oblongata, VP – ventral pallidum, OCh – optic chiasm, 3V – 3rd ventricle, Cx – cortex, CPu – caudate putamen.


  5. Dissociation and plating

    1. Transfer dissected hypothalami into a 15-ml Falcon tube, remove remaining HBSS and add 5 ml digestion solution.

    2. Digest the tissue pieces for 30-60 min at 37 °C while stirring gently every 4-5 min.

      Note: A longer digestion period may increase cell yield, but it will also decrease viability. The incubation time must be determined empirically. Start with 30 min, determine yield and viability as described in Step E13. Increase incubation time if necessary.

    3. Triturate 13 times with a 10-ml serological pipette.

    4. Carefully and slowly triturate 13 times with a fire-polished glass pipette. Avoid creating bubbles during trituration.

    5. Wait 2 min for remaining undissociated tissue to settle and transfer supernatant into a new 15- ml Falcon tube.

    6. Centrifuge at 300 × g for 5 min.

    7. Remove the clear supernatant and resuspend the cell pellet with 3 ml resuspension medium.

    8. Gently triturate 7 times with a fire-polished glass pipette.

    9. Remove remaining tissue clumps by using a 70 µm cell strainer.

    10. Carefully and slowly transfer the cell suspension to 5 ml ovo/albumin inhibition solution in a 15-ml Falcon tube and centrifuge at 70 × g for 5 min to prepare a discontinuous density gradient.

    11. Remove supernatant.

    12. Add 2-3 ml plating medium and resuspend 7 times with a fire-polished glass pipette.

    13. Quantify the number of viable cells by trypan blue exclusion assay (e.g., with a Neubauer chamber).

    14. Seed 3.25 × 105 viable cells/cm2 in plating medium into a 96-well plate double coated with PDL and laminin.


  6. Feeding and lentiviral transduction with Bmal1-luciferase reporter

    1. On the next day, transduce the cells with Bmal1-luciferase lentivirus (Brown et al., 2005). Details for lentiviral particle production are described in Tsang et al. (2020).

      Note: It is recommended to use a GFP-expressing control virus to determine transduction efficiency.

    2. Thaw lentiviral aliquots at room temperature immediately before use. Avoid keeping them for prolonged times at ambient temperature and avoid unnecessary freeze-thaw cycles.

    3. Prepare several lentiviral dilutions (e.g., 0, 1:5, 1:50. 1:250) in feeding medium containing 16 µg/ml polybrene.

      Note: It is recommended testing at least three different concentrations to determine the optimal transduction conditions. We transduced the cells with ~ 1 × 108 infection units (IFUs) per 1 ml in the presence of 8 µg/ml polybrene.

    4. Replace half of the volume of the plating medium by half of the volume of lentiviral particle-containing feeding medium.

    5. 24 h later, refresh half of the volume of the old medium with fresh feeding medium containing 5 µM AraC.

    6. Feed the cells every 3 d with feeding medium, as above, but without AraC.

      Note: It is recommended to perform quality control experiments to ensure that the prepared culture is free from microbial contamination (e.g., bacteria, fungi, or mycoplasma). Mycoplasma contamination can be tested by using PCR-based detection kits (LookOut Mycoplasma PCR Detection Kit, MilliporeSigma). Although cell identification and integrity tests are commonly performed in cell culture, they are not necessary for these primary cultures, since isolated cells are directly used for the experiment and not maintained for longtime.


  7. Synchronization and bioluminescence measurements

    1. On day 9 in vitro (DIV9), synchronize the cells for 2 h with 100 nM dexamethasone. Therefore, pipette 25 µl pre-warmed feeding medium containing 900 nM dexamethasone into the wells containing 200 µl medium and incubate for 2 h at 37 °C and 5% CO2.

    2. During incubation prepare the feeding medium containing 0.5 mM D-luciferin and place it into a water bath at 37 °C.

      Note: D-luciferin is light sensitive. Protect from light.

    3. After incubation aspirate medium and change to pre-warmed feeding medium with 0.5 mM D-luciferin.

    4. Seal the plate with transparent adhesive foil, place it into the microplate reader and start the measurement.

    5. Perform the luminescence measurement without filter at 34 °C with an integration time of 1 min per well.

    6. Normalize all bioluminescence traces by subtracting the 24-h running average and analyze circadian parameters as described previously (Landgraf et al., 2015).

      Note: Synaptic formation starts to be evident at DIV7 (Figure 2). Neurons are considered to be mature at DIV14 (Biffi et al., 2013; Kos et al., 2016). We use DIV9 neurons for standard circadian luciferase experiments. Bmal1-luciferase reporter rhythms are stable for a week without further medium refreshing during the recording.



Figure 2. Synaptic connectivity increases with days in vitro. Representative bright-field (left) and fluorescence (right) images of primary hypothalamic neurons transduced with a GFP-expressing lentivirus. 10× and 20× magnification.

Data analysis

The Mikrowin2000 software allows real-time monitoring of bioluminescence signals. Bmal1-luciferase rhythms are plotted and displayed. During the measurement, wells of interest can be selected and examined. Mikrowin2000 continuously saves the experiment as a *.dat file, which can be opened by the software once the measurement procedure has been completed. To analyze the data, export the raw data to Excel. Calculate the 24-h running average and subtract that from baseline readings for normalization (Figure 3). We use GraphPad Prism for plotting, sine wave fitting, rhythm parameter determination and statistical analyses. Statistical assessment of rhythmicity is done with JTK_cycle or CircaCompare (Hughes et al., 2010; Parsons et al., 2020).



Figure 3. Bioluminescence measurements of synchronized primary hypothalamic neurons expressing Bmal1-luciferase. Bioluminescence traces are normalized by subtracting their 24-h running average. Circadian parameters, such as amplitude, period (wavelength), and dampening rate (K) are determined by fitting a damped sine wave function (Y = Amplitude*exp(-K*X)*sin((2*pi*X/Wavelength) + Phase shift). A. Representative raw data (black) and the calculated 24-h running average (red). B. Representative normalized data (black) and damped sine wave fit (blue). C. Quantification of period, amplitude and dampening rate. Data are presented as mean ± SEM (n = 6).

Acknowledgments

This study was supported by funds of the German Research Foundation (DFG; OS353-7/1, OS353-10/1 and GRK-1957). The protocol was used for Tsang et al. (2020).

Competing interests

The authors report no conflict of interest.

Ethics

Animal experiments reported in this protocol have been approved by the ethics commission of the Ministry of Energy Change, Agriculture, Environment and Digitalization (MELUR) of the State of Schleswig-Holstein (Az 4_2019-10-01_Oster; 2019-2021).

References

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简介

[摘要]内源性生物钟系统使生物能够适应与时间相关的环境变化。结果,大多数生理过程表现出例如能量代谢,免疫功能,睡眠或激素产生的每日节律。下丘脑生物钟已被确认在协调许多这些过程中起特定作用。 原代神经元文化被广泛用作研究神经元内分子事件的生理相关模型。然而,由于昼夜节律包括较长时间范围内的动态分子变化,而这种变化在各个细胞之间会有所不同,因此纵向测量方法对于研究下丘脑神经元昼夜节律的调节至关重要。在这里,我们提供了用于生成表达昼夜节律性荧光素酶报道基因的下丘脑神经元文化的协议。通过执行生物发光测量,此类报告细胞可用于以高时间分辨率纵向监测细胞昼夜节律。


[背景]为了适应重复在其环境中的时间-日期依赖性变化,许多生物已开发出一种内源性生物钟系统调节行为和生理过程的24小时的节律(夏尔马,2003)。在哺乳动物中,一个昼夜节律性起搏器主要位于下丘脑上视交叉上核(SCN)。它与外部时间协调整个身体的细胞时钟调节。睡眠,食欲和新陈代谢的每日模式由下丘脑神经元中的细胞昼夜节律调节(Cedernaes等,2019)。

在哺乳动物细胞中,昼夜节律时钟由互锁的转录-翻译反馈环(TTFL)组成。在核心TTFL中,转录因子昼夜运动输出周期kaput(CLOCK)和脑和肌肉芳基碳氢化合物受体核转运蛋白样蛋白1(BMAL1或ARNTL)激活其自身阻遏物,周期(PER1-3)和隐色蛋白的表达(CRY1 / 2),其导致基因表达的昼夜节律振荡和蛋白质产物的丰度(Ko and Takahashi,2006)。

直接定量mRNA和蛋白质水平的方法,例如定量PCR或Western blotting,需要在不同时间点从不同的个体或制备物中重复采样,这是劳动密集型的,并且在时间间隔短或细胞材料量少的情况下,非常不切实际(Yu和Hardin,2007年)。为了克服这些问题,已经开发了昼夜报道者,其中萤火虫荧光素酶的表达受时钟基因启动子(例如,Bmal1或Per2 )的控制(Brown等,2005;Ramanathan等, 2012;方(Fang)等人,2017)。它们可以通过执行生物发光测量来实时跟踪细胞的昼夜节律,因此被广泛用于研究昼夜节律功能(Ramanathan等人,2012)。

永生化细胞系是分子生物学研究的重要工具,因为它们容易获得,并且可以不受限制地进行扩展(Pan等,2009)。近年来,它们已成为研究下丘脑钟的体外模型(Fick等,2010和2011; Tsang等,2020)。然而,由于永生化细胞系在遗传和表型上与它们的组织起源不同,因此必须严格解释从此类系统获得的数据,并建议使用更具生理相关性的模型系统进行验证(Pan等,2009)。

原代神经元培养物直接从动物的中枢神经系统获得,并保持其组织起源的许多生理和生化特征(Gordon等,2013;Verma等,2020)。因此,它们是解决生理相关性的有用模型系统。在这里,我们提供了一个昼夜节律的Bmal1-荧光素酶报告基因稳定转导的初级下丘脑神经元培养物的产生方法。通过创建这些报告细胞,可以实时观察和定量描述下丘脑神经节律,从而避免了劳动强度大且材料消耗大的生化实验。这些细胞可进一步用于研究各种因素(如激素或代谢产物)的时钟重置效应。

关键字:原发性下丘脑神经元, 昼夜节律生物钟, 萤光素酶报告基因, Bmal1, 昼夜节律


材料和试剂
96孔板(Corning,目录号:3610)
BD Falcon细胞过滤器,70 µm(BD Biosciences,目录号:352350)
100毫米培养皿
35毫米碟
15毫升猎鹰管
10毫升血清移液器
抛光玻璃移液器
2-8个月大的雄性和雌性C57BL / 6J小鼠
Bmal1-萤光素酶慢病毒(Brown等,2005)
聚-D-赖氨酸氢溴酸盐(Millipore Sigma,目录号:P6407)
层粘连蛋白(BD Biosciences,目录号:354232)
汉克的平衡盐溶液HBSS(PAA,目录号:H15-008)
Earle的平衡盐溶液EBSS,带有酚红(Thermo Fisher Scientific,Gibco ,目录号:24010043)
木瓜悬浮液(沃辛顿生物化学公司,目录号:LS003126)
脱氧核糖核酸酶I,DNaseI (沃辛顿生物化学公司,目录号:LS002058)
神经基础培养基,减去酚红(Thermo Fisher Scientific,Gibco ,目录号:12348017)
B-27补充品(Thermo Fisher Scientific,Gibco ,目录号:17504044)
GlutaMAX补充剂(Thermo Fisher Scientific,Gibco ,目录号:35050061)
卵类蛋白(沃辛顿生物化学公司,目录号:LS003085)
牛血清白蛋白(MilliporeSigma ,目录号:A7030)
L-半胱氨酸(MilliporeSigma ,目录号:C7352)
胎牛血清,FBS(Thermo Fisher Scientific,Gibco ,目录号:10500-064)
台盼蓝溶液(MilliporeSigma ,目录号:T8154)
胞嘧啶β -D-阿拉伯呋喃糖苷,AraC (Millipore Sigma,目录号:C1768)
巴斯德移液器(Th。Geyer,目录号:7691061)
2-巯基乙醇(Millipore Sigma目录号:M3148)
胶粘剂透明PCR封条(Biozym ,目录号:600208)
D-萤光素钠盐(Applichem ,目录号:A1006)
聚凝胺,溴化己二甲铵溴化物(Millipore公司Sigma,目录号:H9268)


设备


无菌层流罩(Thermo Fisher Scientific,型号:MSC-Advantage,目录号:51025411 )
多模式酶标仪(Berthold Technologies,型号:Tristar LB941)
˚F升uorescence显微镜(Nikon,型号:Eclipse的Ts2R)
LED单元(尼康,型号:C-LEDFL470)
荧光滤光片立方体(Nikon,型号:C-LED470,激发:470/40 nm,二向色性:500 nm,发射:535/55 nm)
10 ×物镜(尼康,型号:CFI Achromat ADL-10 × ,NA:0.4)
20 ×物镜(尼康,型号:CFI消色差LWD ADL-20 × ,NA:0.4)
血细胞计数器(Laboroptik ,型号:Neubauer )
水浴(GFL,型号:1002)
解剖显微镜(Leica,型号:MZ6)
解剖剪刀(精细科学工具,目录号:91402-14)
解剖钳(精细科学工具,目录号:11000-12)
11号手术刀(羽毛,目录号:200210011)
5号锋利钳(Fine Science Tools,目录号:11252-20)
2 ×弯钳(Fine Science Tools,目录号:11274-20)
学生精细剪刀(精细科学工具,目录号:91460-11)






软件


MicroWin 2000(Labsis ,https : //labsis.de )


程序


安排交配
              要生成E15-16胚胎,请在解剖前安排成年小鼠的交配日15-16 d。我们使用2-8个月大的成年C57BL / 6J小鼠进行交配。
在第二天早晨,通过阴道栓塞检查确认成功交配,如前所述(Behringer等,2016)。
解剖前通过心or或肉眼确认怀孕。


试剂准备
注意:避免对不稳定的试剂重复进行冻融循环。


准备聚-D-赖氨酸(PDL)储备溶液(500 µg / ml)。分成5 ml等分试样,并在-20°C下储存。
准备在PBS中的层粘连蛋白储备溶液(0.1微克/微升)。分成250微升等分试样,并在-20°C下储存。
准备卵/白蛋白抑制溶液,在6 ml EBSS中含有6 mg卵粘液和6 mg牛血清白蛋白。储存在4°C直到使用。
用500 µl EBSS中的1,000 U DNase I制备DNaseI溶液,并储存在-20°C下。
准备平板培养基(Neurobasal + 2%(v / v)B27 + 2 mM Glutamax + 10%(v / v)FBS + 1 ×青霉素/链霉素)并储存在4°C直至使用。将其添加到培养液中之前,在37°C平衡。
准备进料介质(与电镀介质相同,但没有FBS),并在4°C下储存直至使用。将其添加到培养液中之前,在37°C平衡。
准备消化液(100 U木瓜蛋白酶,5 ml EBSS,1.1 mM EDTA(11 µl 0.5 M EDTA),5.5 mM半胱氨酸(3.3 mg),0.067 mM 2-巯基乙醇(2.34 µl 1%(v / v)/14.3 M纯液)和250微升DNA酶I溶液),无菌滤液用0.2 -微米的过滤器。使用前,在37°C下激活20-30分钟。 
准备重悬培养基(2.7 ml EBSS,300 µl卵/白蛋白抑制溶液,150 µl DNaseI溶液)。


PDL和层粘连蛋白双涂层
解剖前一天,用PDL和层粘连蛋白包被96孔板。
用PBS稀释PDL储备液至工作浓度50 µg / ml。将0.5 ml PDL储备溶液(500 µg / ml)与4.5 ml PBS混合以制备5 ml稀释的PDL溶液(50 µg / ml)。
使用前用0.2 µm过滤器过滤。
用7.5 µg / cm 2 PDL覆盖96孔板的培养表面。用48 µl稀释的PDL溶液(50 µg / ml)覆盖96孔板的孔。
在室温下孵育过夜(或在37°C至少孵育2小时)。
用100 µl无菌H 2 O洗涤3次。
允许在无菌细胞培养罩中完全干燥。
在2-8°C下缓慢解冻层粘连蛋白储备溶液。
用PBS稀释层粘连蛋白原液至工作浓度为6.4 µg / ml。将320 µl层粘连蛋白原液(0.1 µg /μl)与4.68 ml PBS混合,以制备5 ml稀释的层粘连蛋白溶液(6.4 µg / ml)。
用50 µl稀释的层粘连蛋白溶液(6.4 µg / ml)覆盖PDL包被的孔。最终涂层浓度约为1 µg / cm 2 。
在37°C下孵育2小时。
用100 µl无菌PBS洗涤3次,添加100 µl平板培养基,并保存在37°C和5%CO 2的培养箱中。


解剖
注意:快速执行的准备过程对于细胞活力至关重要。在准备实验样品之前,请考虑多次实践该程序。在开始解剖之前,请确保已准备好所需的材料并已对所有设备进行了消毒。


使用怀孕母亲的15至16天(E15-16)胚胎(C57BL / 6J母亲通常有8-10头幼仔)。
在层流通风橱下工作,并始终采用无菌技术以减少被细菌,真菌和支原体污染的风险。  
牺牲母亲通过颈椎脱位,用解剖剪刀和镊子打开腹腔。胚胎位于腹腔的后部。
用两个弯曲的镊子小心地将子宫角移开,并轻轻地向相反方向拉动,然后将它们转移到装有冰冷HBSS的100毫米培养皿中。
用解剖剪刀和弯曲镊子提取胚胎,然后将它们转移到装有冰冷HBSS的新的100毫米培养皿中。
用解剖剪刀将胚胎脱垂。
通过将5号尖锐的镊子刺入眼眶并向下压大脑的前额部分,将头部保持在适当的位置。同时使用弯曲的镊子,通过将f从尾到头皮轻轻剥离,以去除外皮和颅骨。从左半球开始,然后从右半球重复该步骤。用弯曲的镊子小心地移开大脑,然后将其放入装有冰冷HBSS的35毫米培养皿中。对其他胚胎重复步骤D5至D7。
注意:拔出大脑时,请避免在组织上施加压力,以保持大脑完整性。此外,注意大脑始终被培养基覆盖,不要让它们变干。


在解剖显微镜下,用镊子和手术刀隔离下丘脑(有关详细信息,请参见图1 )。将它们收集在带有冰冷HBSS的35毫米培养皿中,并放在冰上。使用切开的1000 µl移液器吸头或宽开口的滴管转移被切开的组织。 






图1 。从完整的E16脑清扫下丘脑的步骤。答:翻转大脑,使腹侧部分朝上。通过在乳头体的后边界进行冠状切口来切除大脑的尾部。从第一个切开约1.5 mm的第二个冠状切口,以除去大脑的延髓部。B.旋转剩余的大脑以达到冠状方向,并确保延髓部朝上。解剖下丘脑组织块:从中线各切两个〜0.5 mm的侧向切口,再向前连合处(ac)切一个腹侧。红色虚线表示切割位置。解剖区域以蓝色突出显示。进一步的缩写:LV–侧脑室,塔尔–丘脑,下丘脑–下丘脑,Teg –盖骨膜,Med –延髓,VP –腹侧苍白球,OCh –视交叉,3V –第三脑室,Cx –皮质,CPu –尾状壳。


解离和电镀
将解剖的下丘脑转移到15 ml的Falcon管中,除去残留的HBSS,然后加入5 ml的消化液。
在37°C下将组织碎片消化30-60分钟,同时每4-5分钟轻轻搅拌一次。
注意:较长的消化时间可能会提高细胞产量,但也会降低活力。孵育时间必须根据经验确定。从30分钟开始,按照步骤E13所述确定产量和生存能力。如有必要,增加孵育时间。


用10毫升血清移液器研磨13次。
用火抛光的玻璃移液器小心缓慢地磨碎13次。避免在研磨过程中产生气泡。 
等待2分钟,使剩下的未分离的组织沉淀下来,然后将上清液转移到新的15 ml Falcon管中。
以300 × g离心5分钟。
除去澄清的上清液,并用3 ml的重悬介质重悬细胞沉淀。
用火抛光的玻璃移液器轻轻研磨7次。
使用70 µm细胞过滤器除去残留的组织块。
小心缓慢地将细胞悬液转移至15 ml Falcon管中的5 ml卵黄蛋白/白蛋白抑制溶液中,并以70 × g离心5分钟以制备不连续的密度梯度。
除去上清液。
加入2-3 ml电镀液,并用火抛光的玻璃移液器重悬7次。
通过台盼蓝排除法(例如,使用Neubauer箱)定量活细胞的数量。
将接种培养基中的3.25 × 10 5个活细胞/ cm 2接种到一个用PDL和层粘连蛋白双重包被的96孔板中。


Bmal1-萤光素酶报告基因的喂养和慢病毒转导
第二天,用Bmal1-荧光素酶慢病毒转导细胞(Brown等,2005)。慢病毒颗粒生产的详细信息在Tsang等(2005年)中进行了描述。(2020年)。
注意:建议使用表达GFP的对照病毒来确定转导效率。


使用前立即在室温下解冻慢病毒等分试样。避免在环境温度下长时间放置它们,并避免不必要的冻融循环。
在含有16 µg / ml聚乙烯的进料培养基中准备几种慢病毒稀释液(例如0、1:5、1:50、1:250)。
注意:建议测试至少三种不同的浓度,以确定最佳的转导条件。我们转导〜1细胞× 10 8感染单位每1ml(IFUs)在8微克/毫升的存在聚凝胺。


用一半的含慢病毒颗粒的进料培养基代替平板培养基的一半。
24小时后,用含有5 µM AraC的新鲜进料培养基刷新旧培养基的一半体积。
如上所述,每3 d用饲养培养基饲养细胞,但不添加AraC 。
注意:建议进行质量控制实验,以确保所制备的培养物不受微生物污染(例如,细菌,真菌或支原体)。支原体污染可以通过使用基于PCR的检测试剂盒(LookOut支原体PCR检测试剂盒,MilliporeSigma )进行检测。尽管细胞鉴定和完整性测试通常在细胞培养中进行,但对于这些原代培养而言,它们并不是必需的,因为分离的细胞可直接用于实验,无需长期维护。


同步和生物发光测量
在体外第9天(DIV9),将细胞与100 nM地塞米松同步2 h 。因此,将装有900 nM地塞米松的25 µl预热加料培养基移入装有200 µl培养基的孔中,并在37°C和5%CO 2下孵育2小时。
在孵育过程中,准备含0.5 mM D-荧光素的进料培养基,并将其放入37°C的水浴中。
注意:D-萤光素对光敏感。避光。


孵育后,吸出培养基,并换成含0.5 mM D-荧光素的预热喂养培养基。
用透明的粘合箔密封板,将其放入酶标仪中并开始测量。
在不使用滤光片的情况下,在34°C的条件下进行发光测量,每孔积分时间为1分钟。
通过减去24小时运行平均值对所有生物发光迹线进行归一化,并如前所述分析昼夜节律参数(Landgraf等,2015)。
注意:在DIV7处,突触的形成开始明显(图2)。神经元被认为在DIV14时已经成熟(Biffi等人,2013; Kos等人,2016)。我们将DIV9神经元用于标准生物钟荧光素酶实验。Bmal1-萤光素酶记者节律稳定了一周


录制过程中无需进一步刷新介质。




图2 。突触的连通性随着体外天数的增加而增加。用表达GFP的慢病毒转导的下丘脑神经元的代表性明场(左)和荧光(右)图像。10倍和20倍放大率。


数据分析


Mikrowin2000软件允许实时监控生物发光信号。绘制并显示Bmal1-荧光素酶的节律。在该测量,感兴趣的井可以选择和检查。Mikrowin2000连续将实验保存为*。dat文件,测量过程完成后即可通过软件打开。要分析数据,请将原始数据导出到Excel。计算24小时运行平均值,并从基线读数中减去该平均值以进行归一化(图3)。我们使用GraphPad Prism进行绘图,正弦波拟合,节奏参数确定和统计分析。使用JTK_cycle或CircaCompare对节奏进行统计评估(Hughes等,2010; Parsons等,2020)。




图3 。表达Bmal1-荧光素酶的同步下丘脑神经元的生物发光测量。生物发光迹线通过减去其24小时运行平均值进行归一化。通过拟合阻尼正弦波函数(Y =振幅* exp (-K * X)* sin((2 * pi * X / Wavelength) )+相移)。A .代表原始数据(黑色)和计算出的24小时平均数(红色)。B.代表归一化数据(黑色)和阻尼正弦波拟合(蓝色)。C.周期,幅度的量化数据以平均值±SEM(n = 6)表示。




致谢


这项研究得到了德国研究基金会(DFG; OS353-7 / 1,OS353-10 / 1和GRK-1957)的资助。该协议被用于Tsang等人。(2020年)。


利益争夺


作者报告没有利益冲突。


伦理


该协议中报告的动物实验已获得石勒苏益格-荷尔斯泰因州能源变化,农业,环境和数字化部(MELUR)的伦理委员会的批准(Az 4_2019-10-01_Oster; 2019-2021)。


参考


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Copyright Schmidt et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Schmidt, C. X., Tsang, A. H. and Oster, H. (2021). Generation of Mouse Primary Hypothalamic Neuronal Cultures for Circadian Bioluminescence Assays . Bio-protocol 11(5): e3944. DOI: 10.21769/BioProtoc.3944.
  2. Tsang, A. H., Koch, C. E., Kiehn, J.-T., Schmidt, C. X. and Oster, H. (2020). An adipokine feedback regulating diurnal food intake rhythms in mice. eLife 9: e55388.
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