这项工作得到了 CMRPG3C1771, CMRPG3C1772, CMRPG3E0631, CMRPG3E0632, CMRPG3E0633) 的支持。

这项研究获得了台湾桃园 Linkuo 昌起劲纪念医院机构审查委员会 (102-2577A3) 的批准。
1. NR1-GFP 表达质粒的制备
<ol><li>将 10 ng 的 NR1-GFP 质粒与100µL 的大肠埃希氏菌 DH5α在无菌1.5 毫升离心管中, 吸管混合均匀地上下4-6 次, 并在冰上孵育20分钟。</li>
	<li>在42° c 的水浴中孵育1分钟的试管, 从水浴中取出管子, 将1毫升的性肉汤 (LB) 培养基放入试管中, 并在37° c 的孵化箱中孵育, 并摇动 1 h。</li>
	<li>将50µL 的混合物放在含有氨苄青霉素 (0.1 毫克/毫升) 的 lb 琼脂板上, 在一夜间孵化箱中在37° c 处孵育 lb 琼脂板。</li>
	<li>使用无菌尖端从隔夜 lb 琼脂板中挑选一个菌落, 并在一个含有5毫升培养基和氨苄西林 (0.1 毫克/毫升) 的细菌培养管中旋转尖端。在恒温箱中孵育37° c 的试管, 隔夜摇动。</li>
	<li>分3毫升的夜间细菌培养成500毫升烧瓶, 其中包含250毫升无菌 LB 培养基和氨苄西林 (0.1 毫克/毫升)。在37° c 的晃动下孵育烧瓶。使用光谱仪定期检查600纳米波长的细菌培养的光学密度 (OD), 直到 OD600 达到 0.6-1.0。 注: 混合800µL 的隔夜细菌培养与200µL 甘油准备甘油库存, 并存储在-80 ° c 下的甘油库存, 以备将来使用。</li>
	<li>250毫升细菌培养制备 NR1-GFP 表达质粒<ol>
<li>通过离心在 6000 x g 收集细胞15分钟在4° c。</li>
		<li>重10毫升悬浮缓冲液中含有核糖核酸的细菌颗粒;漩涡彻底, 直到没有看到丛。</li>
		<li>在细菌悬浮液中加入10毫升裂解缓冲液, 轻轻倒置混合物4-6 次, 室温 (RT) 孵育裂解物5分钟。</li>
		<li>将10毫升 pre-chilled 沉淀缓冲液添加到裂解物中, 轻轻翻转混合物4-6 次。</li>
		<li>将裂解液倒入带盖的过滤筒的桶中;在 RT 中等待10分钟。</li>
		<li>卸下墨盒插座喷嘴的盖子, 将柱塞插入墨盒, 然后轻轻按下柱塞。将过滤的裂解液收集到50毫升的管子中。</li>
		<li>添加2.5 毫升专有缓冲从制造商到过滤的裂解物, 混合混合物通过倒置管大约10次, 并且在冰孵育混合物 30 min。</li>
		<li>将过滤过的裂解混合物应用到 pre-equilibrated 10 mL 平衡缓冲器的过滤器柱上。允许柱在重力作用下排出。</li>
		<li>用30毫升洗涤缓冲器两次洗涤柱, 然后用15毫升洗脱缓冲器从柱洗 DNA。</li>
		<li>将10.5 毫升 (0.7 卷) 异丙醇添加到洗 DNA 缓冲液中, 混合均匀, 并将混合物离心 2万 x g, 在4° c 处为30分钟。</li>
		<li>仔细醒酒上清液, 用5毫升内毒素-70% 乙醇一次性清洗 DNA 颗粒, 在 2万 x 克上离心10分钟。</li>
		<li>醒酒上清液, 在化学罩中干燥5-10 分钟的颗粒, 并在300-500 µL 内毒素释放缓冲剂中溶解颗粒。</li>
		<li>用分光光度计测定紫外260/280 纳米溶液的吸收量, 确定质粒浓度。 注: 使用同一协议制备表达质粒 GFP。</li>
	</ol>
</li>
</ol>2. NR1-GFP 表达质粒转染 HEK293 细胞
<ol><li>分200µL 2% 明胶溶液对48井培养板的每一个井, 并在37° c 下孵育至少30分钟的板材。</li>
	<li>从井中抽出明胶溶液, 并在每井的200µL 中含有10% 胎牛血清 (FBS) 的细胞培养基中, 5 x 104 HEK293 细胞。在37° c 的湿润恒温箱中孵育盘, 5% CO2过夜。 注意: 使用例计数单元格。</li>
	<li>第二天, 用200µL 的新鲜培养基取代含有 10% FBS 的培养基。</li>
	<li>为每一个井, 准备溶液 A 混合 100 ng NR1-tGFP 质粒与20µL 培养基, 和溶液 B 混合0.8 µL 转染试剂与20µL 培养基。将每个实验的总容积所需的井数乘以。</li>
	<li>通过混合溶液 a 和溶液 B 制备溶液 C, 并在 RT 处孵育20-30 分钟的混合物。</li>
	<li>分40µL 的溶液 C 的每一个井含有 HEK293 细胞, 轻轻地旋转板, 并孵化在37° c 在一个湿化孵化器提供 5% CO2过夜的板块。</li>
	<li>在荧光显微镜下检查 NR1-GFP 重组蛋白在 40 x 200 x 放大 (激发/发射: 482/502 nm) 下的表达, 并进行细胞免疫荧光检测。 注: 染表达质粒 GFP 进入 HEK293 细胞使用相同的协议。</li>
</ol>3. 细胞免疫荧光法
<ol><li>从水井中抽出所用的培养基, 然后用200µL 磷酸缓冲盐水 (PBS) 三次冲洗每一个井。</li>
	<li>每井加200µL 4% 甲醛溶液;在 RT 上孵育板15分钟。</li>
	<li>从井中吸取甲醛溶液, 用200µL PBS 三次冲洗每口井。</li>
	<li>在 PBST (PBS 的 0.1% Tween-20) 溶液中加入200µL 10% 脱脂牛奶, 并在 RT 中孵育 1 h, 轻轻摇晃。</li>
	<li>从井中抽出 PBST 溶液中的10% 脱脂牛奶, 然后用200µL PBST 冲洗一次。</li>
	<li>用稀释过的等离子体 (1:100 在 PBST) 孵育水井, 作为在 RT 1 小时内温和摇动的主要抗体。 注: 血浆是从疑似有自身免疫性脑炎的患者血液中获得的。</li>
	<li>从水井中吸取稀释后的等离子体, 用200µL PBST 三次洗涤每一个井。</li>
	<li>添加200µL 山羊人 IgG 共轭与 Alexa 594 (1:1, 000 稀释 PBST) 每井, 并孵化培养板在 RT 与温和晃动 1 h。</li>
	<li>吸取山羊人 IgG 共轭的 Alexa 594 溶液, 并洗涤每井与200µL 的 PBST 三倍。</li>
	<li>添加100µL 甘油溶液 (PBS 50% 甘油和 1:100,000 4 ", 6-diamidino-2-吲 (DAPI)) 每井。</li>
	<li>使用10X 目镜、4X、10X 和20X 的物镜观察和成像荧光显微镜下的细胞。 注意: 为每染料使用正确的过滤器: 为 NR1-GFP (励磁或放射 = 482/502 毫微米), 为 Alexa 流体 594 (励磁或放射 = 561/594 毫微米), 为 DAPI (励磁或放射 = 358/461 毫微米)。用同样的方法对 HEK 细胞进行负控制。</li>
</ol>

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作者没有什么可透露的。

有几种细胞免疫分析筛选的存在自身抗体对 NMDA 受体的文献报道, 包括活细胞免疫荧光法检测11, 固定细胞免疫荧光检测9和流 cytometry-based 检测14。在 ectopically 表达 NR1 蛋白的细胞制备后不久, 应进行活体细胞免疫荧光检测, 而流 cytometry-based 检测则需要训练有素的人员和专用设备。固定细胞免疫荧光法是一种更方便的化验, 可以进行临床设置。因...

抗 NMDA 受体自身免疫性脑炎是一种新的公认的疾病实体, 可以发生在所有年龄的患者, 并影响主要女性患者1,2。它是临床上最常见的脑炎之一, 最初不明病因的脑炎3。感染抗 NMDA 受体脑炎的患者通常有前驱症状的头痛或发烧, 其次是意识水平的快速发展变化和各种急性神经精神症状, 包括躁动, 烦躁, 焦虑, 失眠, 幻觉, 妄想, 侵略, 怪异行为, 运动异常, 自主失调, 和癫痫发作4,5。早期认识到这种情况和及时治疗的免疫疗法是重要的结果, 甚至完全恢复在受影响的患者6。因此, 建议抗 NMDA 受体自身免疫性脑炎应被认为是一个重要的鉴别诊断病人呈现急性或新发精神病功能7,8。
除临床特点外, 检测血液或脑脊液中 nmda 受体的自身抗体, 对于准确诊断抗 nmda 受体自身免疫性脑炎是必不可少的9。大多数检测抗 NMDA 受体自身抗体的免疫学试验在几个研究实验室中都是可用的10,11, 并且只有一个商业可用的细胞免疫荧光检测方法来筛选抗 NMDA 受体抗体12。本研究的目的是开发一种简单的 in-house 细胞免疫荧光分析法, 可以方便地用于实验室筛选抗 nmda 受体自身抗体的存在, 促进抗 nmda 受体的临床研究自身免疫性脑炎。NMDA 受体是一种 heterotetramer 的离子通道蛋白复合体, 在大脑中明确表达。它由两个强制 NR1 子单元组成, 以及 NR2A、NR2B、NR2C 或 NR2D13的一个或两个子单元的组合。先前的一项研究报告, 抗体靶向的主要表位位于 NR1 亚基5的胞外 N 端域。因此, 在本协议中, 我们表达了人类胚胎肾上皮细胞系 (HEK293) 中标记有绿色荧光蛋白 (GFP) 的 NMDA 受体的人重组 NR1-subunit 蛋白, 并建立细胞免疫荧光法检测血液中抗 NMDA 受体自身抗体的 IgG 类。

<table>
	<tbody>
		<tr>
			<td>GRIN1 (GFP-tagged) - Human glutamate receptor, ionotropic, N-methyl D-aspartate 1 (GRIN1), transcript variant NR1-1</td>
			<td>Origene (Rockville, MD, USA)</td>
			<td>RG219368</td>
			<td>NR1-cDNA clone tagged with C-terminal tGFP sequences</td>
		</tr>
		<tr>
			<td>pCMV6-AC-GFP</td>
			<td>Origene (Rockville, MD, USA)</td>
			<td>PS100010</td>
			<td>mammalian vector with C-terminal tGFP tag</td>
		</tr>
		<tr>
			<td>EndoFree Plasmid Maxi Kit</td>
			<td>Qiagen (Hilden Germany)</td>
			<td>12362</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000 Transfection Reagent</td>
			<td>Invitrogen (Carlsbad, CA, USA)</td>
			<td>11668-019</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM I Reduced Serum Medium</td>
			<td>Thermo Fisher</td>
			<td>31985-070</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, High Glucose, Pyruvate</td>
			<td>Thermo Fisher</td>
			<td>11995-065</td>
			<td></td>
		</tr>
		<tr>
			<td>Characterized Fetal Bovine Serum, US Origin</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SH30071.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Human IgG (H+L) Secondary Antibody, Alexa Fluor 594 conjugate</td>
			<td>Thermo Fisher</td>
			<td>A-11014</td>
			<td></td>
		</tr>
		<tr>
			<td>Positive control: anti-glutamate receptor (type NMDA)</td>
			<td>Euroimmun AG, L&#252;beck, Germany</td>
			<td>CA 112d-0101</td>
			<td></td>
		</tr>
		<tr>
			<td>Euroimmun Assay Kit</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a simple cell-based immunofluorescence assay to detect autoantibody against the n-methyl-d-aspartate (nmda) receptor in blood

抗 nmda 受体自身抗体的存在可导致各种神经精神症状的患者, 称为抗 nmda 受体自身免疫性脑炎。对血液或脑脊液中 NMDA 受体的特定自身抗体的检测是准确诊断这一病症的必要条件。nmda 受体是一个离子通道蛋白复合体, 包含四亚基, 包括两个强制性 nmda 受体亚基 1 (NR1) 和一个或两个 nmda 受体亚基 2A (NR2A), nmda 受体亚基 2B (NR2B), nmda 受体亚基 2C (NR2C), 或 nmda 受体亚基 2D (NR2D)。报道了 nmda 受体 NR1 亚单位的胞外 N 端区存在抗 nmda 受体自身抗体的表位。本研究的目的是开发一种简单的细胞免疫荧光检测方法, 可作为筛选试验, 检测血液中 NMDA 受体 NR1 亚基自身抗体的存在, 以促进临床和基础研究抗 NMDA 受体自身免疫性脑炎。

根据本议定书1节所述的程序, 平均可从250毫升细菌培养中获得200-300 µg 内毒素-游离表达质粒 NR1-GFP 和 GFP。本议定书2节所述的 48-井板中培养的 HEK293 细胞的转染量为 100 ng/很好的表达质粒。24-30 h 在转染后, 细胞表达 NR1-GFP, 荧光显微镜下可检测 GFP 重组蛋白。图 1A显示了表达 NR1-GFP 的宿主单元格的图像, 而图 1D显示了表达 GFP 的单...

Watch this Scientific Journal Video about A Simple Cell-based Immunofluorescence Assay to Detect Autoantibody Against the N-Methyl-D-Aspartate NMDA Receptor in Blood at JoVE.com

A Simple Cell-based Immunofluorescence Assay to Detect Autoantibody Against the N-Methyl-D-Aspartate NMDA Receptor in Blood

我们 ectopically 表达了以绿色荧光蛋白标记的 nmda 受体在人胚细胞 (HEK293) 中的 NR1 亚基作为抗原, 以检测疑似自身免疫性脑炎患者血液中 nmda 受体的自身抗体。这种简单的方法可能适用于临床设置的筛选目的。

血液中 n-甲基-d-天门冬氨酸 (NMDA) 受体自身抗体的简易细胞免疫荧光检测

The presence of anti-NMDA receptor autoantibody can cause various neuropsychiatric symptoms in the affected patients, termed anti-NMDA receptor autoimmune ...

a-simple-cell-based-immunofluorescence-assay-to-detect-autoantibody

Research

JoVE Journal

Neuroscience

A Simple Cell-based Immunofluorescence Assay to Detect Autoantibody Against the N-Methyl-D-Aspartate (NMDA) Receptor in Blood

9.8K 次观看.  Chang Gung Memorial Hospital-Linkou. 我们 ectopically 表达了以绿色荧光蛋白标记的 nmda 受体在人胚细胞 (HEK293) 中的 NR1 亚基作为抗原, 以检测疑似自身免疫性脑炎患者血液中 nmda 受体的自身抗体。这种简单的方法可能适用于临床设置的筛选目的。

视频: A Simple Cell-based Immunofluorescence Assay to Detect Autoantibody Against the N-Methyl-D-Aspartate NMDA Receptor in Blood

Application of a NMDA Receptor Conductance in Rat Midbrain Dopaminergic Neurons Using the Dynamic Clamp Technique

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the electrical activity of one or more neurons in response to an experimentally-controlled input. The electrical activity of neurons can be recorded in isolated brain slices using patch clamp techniques with glass micropipettes. Traditionally, experimenters can mimic neuronal input by direct injection of current through the pipette, electrical stimulation of the other cells or remaining axonal connections in the slice, or pharmacological manipulation by receptors located on the neuronal membrane of the recorded cell.
Direct current injection has the advantages of passing a predetermined current waveform with high temporal precision at the site of the recording (usually the soma). However, it does not change the resistance of the neuronal membrane as no ion channels are physically opened. Current injection usually employs rectangular pulses and thus does not model the kinetics of ion channels. Finally, current injection cannot mimic the chemical changes in the cell that occurs with the opening of ion channels.
Receptors can be physically activated by electrical or pharmacological stimulation. The experimenter has good temporal precision of receptor activation with electrical stimulation of the slice. However, there is limited spatial precision of receptor activation and the exact nature of what is activated upon stimulation is unknown. This latter problem can be partially alleviated by specific pharmacological agents. Unfortunately, the time course of activation of pharmacological agents is typically slow and the spatial precision of inputs onto the recorded cell is unknown.
The dynamic clamp technique allows an experimenter to change the current passed directly into the cell based on real-time feedback of the membrane potential of the cell (Robinson and Kawai 1993, Sharp et al., 1993a,b; for review, see Prinz et al. 2004). This allows an experimenter to mimic the electrical changes that occur at the site of the recording in response to activation of a receptor. Real-time changes in applied current are determined by a mathematical equation implemented in hardware.
We have recently used the dynamic clamp technique to investigate the generation of bursts of action potentials by phasic activation of NMDA receptors in dopaminergic neurons of the substantia nigra pars compacta (Deister et al., 2009; Lobb et al., 2010). In this video, we demonstrate the procedures needed to apply a NMDA receptor conductance into a dopaminergic neuron.

In this video, we demonstrate how to apply a conductance into a dopaminergic neuron recorded in the whole cell configuration in rat brain slices. This technique is called the dynamic clamp.

一个NMDA受体电导的大鼠中脑多巴胺能神经元的动态钳技术中的应用

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the ...

科研

神经科学

A Simple Way to Measure Ethanol Sensitivity in Flies

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain is correlated with that strain s ethanol preference, and therefore sedation is a highly relevant measure to study the genetics of alcohol responses and drinking. We demonstrate a simple way to expose flies to ethanol and measure its intoxicating effects. The assay we describe can determine acute sensitivity, as well as ethanol tolerance induced by repeat exposure. It does not require a technically involved setup, and can therefore be applied in any laboratory with basic fly culture tools.

A simple assay to measure the sedating effects of ethanol on Drosophila flies, based on the loss of righting reflex, is described.

一个简单的方法来衡量在果蝇的乙醇灵敏度

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain ...

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

成像pHluorin标记插入到质膜受体原代培养的小鼠神经元

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once.

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Xenopus laevis tadpoles prefer swimming on the white side of a black/white tank. This behavior is guided by their vision. Based on this behavior, we present a simple assay to test the visual function of tadpoles.

一个简单的行为分析在检测视觉功能<em&gt;非洲爪蟾</em

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

测定冷灵敏度和适应小鼠一种简单廉价的方法

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

一个简单的方法来衡量在改建悬赏寻求行为使用<em&gt;果蝇</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

一个简单的一步解剖协议成人全安装准备<em&gt;果蝇</em&gt;脑

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by immunizing mice with neurogenic peptides to direct an inflammatory attack toward components of the peripheral nervous system (PNS). Recent advances have enabled the induction of EAN in the relatively resistant C57BL/6 mouse line using myelin protein zero (P0)106-125 or P0180-199 peptides delivered in adjuvant combined with the injection of pertussis toxin. The ability to induce EAN in the C57BL/6 strain allows for the use of the numerous genetic tools that exist on this mouse background, and thus allows the sophisticated study of disease pathogenesis and interrogation of the mechanistic action of novel therapeutics in combination with transgenic approaches. In this study, we demonstrate a simple approach to successfully induce EAN using the P0180-199 peptide in C57BL/6 mice. We also outline a protocol for the assessment of functional deficits that occur in this model, accompanied by an array of neuropathological features. Thus, this model is a powerful experimental model to study the pathogenesis of human peripheral demyelinating neuropathies, and to determine the efficacy of potential therapies that aim to promote myelin repair and protect against nerve damage in autoimmune neuritis.

This report outlines a simple approach to successfully induce experimental autoimmune neuritis (EAN) using the myelin protein zero (P0)180-199 peptide in combination with Freund&#39;s complete adjuvant and pertussis toxin. We present a sophisticated paradigm capable of accurately assessing the extent of functional deficits and neuropathology that occur in this EAN.

C57BL/6 小鼠自身免疫性神经炎的功能和病理评价的简易方法

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.

创新 RNA原位检测技术在 D1-Alpha 鼠伏隔核中多巴胺受体的鉴定

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in ...

A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR malfunction, expressed as either over- or under-activity caused by mutations, altered expression, trafficking, or localization, can contribute to numerous diseases, especially in the central nervous system. Therefore, understanding the receptor&#39;s biology as well as facilitating the discovery of compounds and small molecules is crucial in ongoing efforts to combat neurological diseases. Current approaches to studying the receptor have limitations including low throughput, high cost, and the inability to study its functional abilities due to the necessary presence of channel blockers to prevent NMDAR-mediated excitotoxicity. Additionally, the existing assay systems are sensitive to stimulation by glutamate only and lack sensitivity to stimulation by glycine, the other co-ligand of the NMDAR. Here, we present the first plate-based assay with high-throughput power to study an NMDA receptor with sensitivity to both co-ligands, glutamate and D-serine/glycine. This approach allows the study of different NMDAR subunit compositions and allows functional studies of the receptor in glycine- and/or glutamate-sensitive modes. Additionally, the method does not require the presence of inhibitors during measurements. The effects of positive and negative allosteric modulators can be detected with this assay and the known pharmacology of NMDAR has been replicated in our system. This technique overcomes the limitations of existing methods and is cost-effective. We believe that this novel technique will accelerate the discovery of therapies for NMDAR-mediated pathologies.

The goal of this protocol is to facilitate the study of NMDA-receptors (NMDAR) at a larger scale and allow the examination of modulatory effects of small molecules and their therapeutic applications.

高通量钙流量测定对甘氨酸/d-丝氨酸和谷氨酸敏感的 NMDA 受体的研究

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR ...