我们提出了一个协议,用于容量神经递质释放的光学检测创建基于细胞的神经递质荧光工程记者(CNiFERs)。
基于细胞的神经递质荧光工程记者(CNiFERs)为神经科学家光学检测神经递质的释放在脑中体内的新工具。具体CNiFER是从稳定地表达特定的G蛋白偶联受体,其耦合至G Q / 11的G蛋白,以及一个基于FRET的钙 -detector,TN-XXL人胚肾细胞产生。该受体的活化导致的增加的FRET信号。 CNiFERs有纳米灵敏度和几秒钟的时间响应,因为一个CNiFER克隆利用天然受体对特定的神经递质, 例如 D2R为多巴胺。 CNiFERs被直接注入到大脑,使他们能够感神经递质释放与小于一百微米的空间分辨率,这使得它们理想来测量体内体积传输。 CNiFERs也可以用来筛选其它药物用于在vi潜在的交叉反应性VO。我们最近扩大CNiFERs的系列,包括G蛋白偶联受体的夫妇至G I / O的G蛋白。 CNiFERs可用于检测乙酰胆碱(ACh),多巴胺(DA)和去甲肾上腺素(NE)。鉴于任何GPCR可用于创建一个新颖CNiFER并有大约在人类基因组800的GPCR,我们在这里描述的一般方法来设计,实现和测试的任何类型的CNiFER的。
为了全面了解神经元在大脑中的通信方式,它必须有用于测量神经递质在体内的释放的方法。有在体内神经递质检测几种行之有效的方法。一种常用的技术是微透析,其中套管被插入到脑和脑脊液中的一小体积被收集并用高效液相色谱法和电化学检测1进行分析。微透析有探针的几个直径的量级的空间分辨率, 例如 ,约0.5毫米的直径200微米的微探针。这种技术的时间分辨率,但是,是缓慢由于采样间隔,通常持续约5分钟以上1。此外,分析还没有实时进行。另一种技术是快速扫描循环伏安(FSCV),其使用插入到大脑的碳纤维探针。 FSCV具有优良的温度口服分辨率(亚秒),高灵敏度(毫微摩尔),并与探针直径的5至30微米的空间分辨率。然而,FSCV被限制为在碳势探头2产生的特性的氧化和与电压降低信息发射机。
测量神经递质第三种技术是直接通过基因编码的神经递质(NT)生物传感器3。用这种方法,融合蛋白创建包含用于耦合到荧光共振能量转移(FRET)系一对荧光团4或置换的GFP 5的发射机的配体结合域。不同于前两种方法,这些生物传感器遗传编码和表达的宿主细胞的表面上,如神经元,通过生产转基因动物的或急性与使用病毒试剂的感染细胞。迄今为止,遗传编码的生物传感器已被被仅用于detectin开发摹谷氨酸和GABA 3-5。这些技术的限制一直是低的灵敏度,在nM范围内,以及无法扩大检测到大量的发送器, 例如 ,经典的神经递质,神经肽和神经调质,其通过G蛋白偶联受体(GPCR)的信号。事实上,也有人类基因组近800 G蛋白偶联受体。
为了解决这些不足,我们已经开发了一种创新工具,通过一个GPCR信号的任何神经递质的光学测量释放。 CNiFERs(基于细胞的神经递质的荧光工程记者)的设计,以表达特定的GPCR是,在受到刺激时,会触发钙离子浓度由遗传编码的基于FRET的钙离子传感器检测到细胞内增加克隆HEK293细胞, TN-XXL。因此,CNiFERs变换神经递质受体结合到荧光变化,提供了一个直接和实时的光读EAD出局部神经递质的活动。通过利用天然受体对于给定的神经递质,CNiFERs保留化学特异性,亲和力和内源性表达的受体的时空动力 学。到目前为止,我们已经创建了三种CNiFERs,一个用于使用M1受体,一个用于检测多巴胺检测乙酰胆碱使用D2受体,和一个用于使用α1A受体6,7-检测去甲肾上腺素。该CNiFER技术很容易扩展和可扩展性,使得它适合于任何类型的GPCR的。在这个朱庇特的文章中,我们描述和示出了在体内 CNiFERs设计,实现的方法,及测试方法的任何应用。
CNiFERs的创建提供了用于体内光学测量大脑神经递质释放的创新和独特的策略。 CNiFERs非常适合用于测量突触外发布, 即音量传导,神经递质。重要的是,每CNiFER具有天然的GPCR的性质,提供了在大脑中的神经递质的水平的变化的生理光学测量。迄今,已被用于检测乙酰创建CNiFERs(M1-CNiFER)6,多巴胺(D2-CNiFER)7和去甲肾上腺素(α1A-CNiFER)7。
<p class="jove_co…The authors have nothing to disclose.
我们感谢B.康克林(美国加州大学旧金山分校),用于提供对G qi5和G QS5的cDNA,A史怀哲寻求帮助,以电子,N.泰勒与克隆,伊恩Glaaser和罗伯特·里夫金的校对筛查援助,和Olivier Griesbeck为TN-XXL。 (; DA037170 DA029706),生物医学成像和生物工程研究所(NIBIB)(EB003832),霍夫曼 – 罗氏(88610A)和“神经科学这项工作是通过药物滥用美国国家研究所(NIDA)的研究资助项目通过NIDA(DA007315)关于滥用“培训资助的药物。
pCDH-CMV-MCS-EF1-Puro | System Biosciences | CD510B-1 | Cloning: for generating lentivirus |
12×75 *BD Falcon High Clarity Polypropylene Round Bottom Test Tube | BD Biosciences | 352063 | FACS |
BD 40 um Falcon cell strainers | BD Biosciences | 352340 | FACS |
0.05% Trypsin EDTA | Invitrogen | 25200056 | FACS |
96 Well Plate, flat bottom, clear | Corning | 3596 | FACS |
96 well cell culture plates | Corning | CLS3997 | Flexstation |
Optilux black clear bottom | Corning | 3603 | Flexstation |
Flexstation pipet tips | Molecular Devices | 9000-0911 | Flexstation |
Acetylcholine Chloride | SimgaAldrich | A2661 | Flexstation |
Norepinephrine | SimgaAldrich | A7256 | Flexstation |
Dopamine Hydrochloride | SimgaAldrich | PHR1090 | Flexstation |
GABA | SimgaAldrich | A2129 | Flexstation |
Histamine | SimgaAldrich | H7125 | Flexstation |
Glutamate | SimgaAldrich | 49621 | Flexstation |
Epinephrine | SimgaAldrich | E4642 | Flexstation |
Somatostatin | SimgaAldrich | S1763 | Flexstation |
5HT | SimgaAldrich | H9523 | Flexstation |
VIP | Alpha Diagnostics Inc. | SP-69627 | Flexstation |
Orexin A | Alpha Diagnostics Inc. | 12-p-01 | Flexstation |
Substance P | SimgaAldrich | S6883 | Flexstation |
Adenosine | SimgaAldrich | A4036 | Flexstation |
Melatonin | SimgaAldrich | M5250C | Flexstation |
Fluorescence Plate Reader & software | Molecular Devices | Flexstation 3 | Flexstation |
DMEM (high glucose) with Glutamax | Life Technologies | 10569-010 | Tissue culture |
Fetal bovine serum | Life Technologies | 10082-139 | Tissue culture |
Pen/Strep | Life Technologies | 15140-122 | Tissue culture |
Puromycin | InvivoGen | ant-pr-1 | Tissue culture |
Fibronectin | SimgaAldrich | F0895 | Tissue culture |
CoolCell LX Alcohol-free controlled-rate cell freezing box | Bioexpress | D-3508) | Tissue culture |
cyanoacrylate glue | Loctite | Loctite no. 495 | surgery and stereotaxic injection |
plastic paraffin film | VWR | Parafilm® | surgery and stereotaxic injection |
NANOINJECTOR | Drummond | 3-000-204 | surgery and stereotaxic injection |
GLASS ELECTRODES | Drummond | 3-000-203G | surgery and stereotaxic injection |
hand held drill | OSADA | Exl-M40 | surgery and stereotaxic injection |
Burrs for drill | Fine Scientific | 19007-05; 19007-07) | surgery and stereotaxic injection |
Sterilizing bath | FST | 18000-45, Hot Bead Sterilizer | surgery and stereotaxic injection |
isoflurane chamber/mask | Highland Medical Equipment | 564-0427, HME 109 Table Top Anesthetic Machine with Isoflurane Vaporizer, O2 Flowmeter, Gang Valve; 564-0852, Induction Chamber 16X7X7.5cm | surgery and stereotaxic injection |
3D scope with arm | Zeiss | surgery and stereotaxic injection | |
fiber optic light | surgery and stereotaxic injection | ||
Betadine | surgery and stereotaxic injection | ||
70 % (v/v) isopropyl alcohol | surgery and stereotaxic injection | ||
Povidone-Iodine Prep Pads | dynarex | 1108 | surgery and stereotaxic injection |
NaCl 0.9% (INJECTION, USP, 918610) | surgery and stereotaxic injection | ||
CYCLOSPORINE (INJECTION, USP) | surgery and stereotaxic injection | ||
Buprenex (INJECTION) buprenorphine (0.03 μg per g rodent) | Sigma | surgery and stereotaxic injection | |
Ophthalmic ointment | Akorn | NDC 17478-235-35 | surgery and stereotaxic injection |
Surgifoam | Ethicon | surgery and stereotaxic injection | |
Grip dental cement | Dentsply | #675571, 675572 | surgery and stereotaxic injection |
Instant SuperGlue | NDindustries | surgery and stereotaxic injection | |
LOCTITE 4041 | surgery and stereotaxic injection | ||
METABOND | C&B | surgery and stereotaxic injection | |
no. 0 cover glass | Fisher | surgery and stereotaxic injection | |
stereotaxic frame | Kopf | surgery and stereotaxic injection | |
Rectal probe and heating pad | FHC | 40-90-8D, DC Temperature Controller,40-90-2-06, 6.5X9.5cm Heating Pad40-90-5D-02, Rectal Thermistor Probe | surgery and stereotaxic injection |
optical breadboard for imaging | Thorlabs | surgery and stereotaxic injection | |
Mineral oil | Fisher | S55667 | surgery and stereotaxic injection |
Kwik-Cast (Silicone elastomer) | World Precision Instruments | surgery and stereotaxic injection | |
Suture | Ethicon | 18’’, 1667, 4-0 | surgery and stereotaxic injection |
Scissors | Fine Scientific Tools | 91500-09, 15018-10 | surgery and stereotaxic injection |
Forcepts | Fine Scientific Tools | 11252-30; #55, 11295-51; Grafe, 11050-10 | surgery and stereotaxic injection |
Student Halsted-Mosquito Hemostats | Fine Scientific Tools | 91308-12 | surgery and stereotaxic injection |
Small Vessel Cauterizer Kit | Fine Scientific Tools | 18000-00 | surgery and stereotaxic injection |
Hot Bead Sterilizers | Fine Scientific Tools | 18000-45 | surgery and stereotaxic injection |
Instrument Case with Silicone Mat | Fine Scientific Tools | 20311-21 | surgery and stereotaxic injection |
Plastic Sterilization Containers with Silicone Mat | Fine Scientific Tools | 20810-01 | surgery and stereotaxic injection |
2P fixed-stage fluorescence scope for in vivo imaging | Olympus | FV1200 MPE | in vivo imaging |
Multiphoton laser | SpectraPhysics | Mai Tai DeepSee | in vivo imaging |
Green Laser | Olympus | 473 nm Laser | in vivo imaging |
xy translation base | Scientifica | MMBP | in vivo imaging |
FRET filter cube for YFP and CFP | Olympus | in vivo imaging | |
25-X water immersion objective | Olympus | in vivo imaging | |
air table | Newport | in vivo imaging | |
custom built light-tight cage | Thorlab | in vivo imaging |