这里所描述的工作是由退伍军人事务部，美国能源部生物医学实验室的研究和开发BX001686-1A1和VA康复研究和开发。

1。电镀细胞培养和维护<ol><li>温暖的Dulbecco改良的Eagle培养基（DMEM）培养基（DMEM +10％FBS +1％青霉素/链霉素/新霉素）至37℃。 </li><li>获得冷冻A12细胞的股价从-80°C。 </li><li>快速解冻细胞37℃水浴;一旦解冻，轻轻吸细胞转移到一个75 厘米通风培养瓶中，并加入10 ml培养基。 </li><li>烧瓶孵育过夜，5％CO 2培养箱在37°C吸介质后24小时，更换新鲜的培养基。持续到70-80％汇合得到（2-3天生长）生长的细胞。 </li></ol> 2，计数和细胞电镀<ol><li>温暖的DMEM和1x-胰蛋白酶-EDTA溶液至37℃。 </li><li>从细胞中吸出物和介质洗2次，用无菌磷酸盐缓冲盐水（PBS）中。 </li><li>加入500μl的胰蛋白酶溶液，并在37℃下2-5分钟。 </li><li>分离用刮刀和中止细胞在5ml的媒体从烧瓶中的细胞。通过过滤器传递细胞并通过细胞过滤到一个干净的50ml管中。可能需要通过过滤器传递细胞不止一次，但不要重复超过三次。 </li><li>使用血细胞计数器确定细胞的量，以培养种子6000细胞/孔在96孔透明底的黑色或白色的围墙板中的100μl的介质的最终体积计数细胞。在5％CO 2下孵育板在37℃下放置24小时。 </li></ol> 3，确定细胞活力和Caspase-3/7活性<ol><li>转移PA到灭菌的5毫升玻璃小瓶中，并溶解在100％二甲亚砜（DMSO）中。稀库存PA溶液1:10 DMSO中，并把它添加到预热的DMEM培养液，以获得最终的工作为0.1mM PA浓度。用于控制媒体，添加DMSO中，添加到含有PA的介质相同的浓度。 </li><li>在各取出介质井和更换50微升媒体+ / -巴勒斯坦权力机构。孵育板2小时，在5％CO 2在37℃下然后加入5微升刃天青试剂后，于室温下10分钟。 </li><li>使用多模酶标仪，记录荧光（560 EX / 590 EM）来衡量细胞活力。结果表示为相对荧光单位（RFU）。 </li><li>到相同的板中，添加55微升蛋白酶试剂和在室温下孵育2小时。 </li><li>再次使用酶标仪，记录发光，从而测量胱天蛋白酶3/7活性。结果表示为相对发光单位（RLU），用发光成正比的胱天蛋白酶3/7活性。 </li><li>正常化的caspase-3/7活性的细胞计数，由胱天蛋白酶3/7活性（RLU）除以细胞活力（RFU）。 </li></ol>

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作者没有冲突披露。

在多重检测是一个已经在许多应用，如PCR芯片，免疫检测，以及其他基于蛋白质的检测方法13，14被科学家广泛接受的技术。最近，复用实验已经越来越利用体外板基础实验和已被确认为评估细胞毒性的准确方法和可行性12。在上述协议中，我们证明了复用荧光灯和发光检测的有效性，以确定在A12单元格下面的PA挑战的caspase-3/7活性和细胞活力。细胞活力，在初始的PA侮辱10分?...

饮食富含饱和脂肪酸如棕榈酸（PA）的已与肥胖和其他并发症，包括心血管疾病和糖尿病1,2。高脂肪饮食也已显示出增加的氧化应激，细胞凋亡，和在下丘脑的神经元变性，食欲和能量消耗3-7中的重要调节器。了解通过高脂饮食诱导的曝光下丘脑功能失调的机制是对肥胖的药物治疗发展重要的。然而，通过这些食物中的脂肪会影响神经元功能的细胞机制仍不清楚。更好地理解如何脂肪如PA可能会引发发病丘脑凋亡途径是迈向这一目标的必要的第一步。本文的目的是描述一个多重检测用于体外测试与PA曝光神经元的反应，对于在H的研究开发利用ypothalamic神经退行性病变。我们提供了一种体外的96孔格式的多重检测，用于测量胱天蛋白酶每个细胞中分化的永生化的成体小鼠的下丘脑细胞与PA的8氧化攻击后线（称为A12）总数3/7活性的详细说明。 简单地说，细胞存活率确定通过基于刃天青分析如下PA的挑战。刃天青是经历酶减少，代谢活跃的细胞的细胞渗透性化合物想到一个过程，通过线粒体9出现。活细胞连续转换刃天青到试卤灵，产生的荧光信号正比于活细胞的数量。胱天蛋白酶3/7活性，随后分析使用DEVD-基于lumogenic测定。 DEVD是由胱天蛋白酶-3切割的氨基酸序列（ASP-GLU-VAL-ASP）。当该序列被耦合到lumogenic物质，在激活时细胞内的胱天蛋白酶3/7的和随后的裂解DEVD基板，发光产物被释放。该反应正比于胱天蛋白酶活性，从而诱导细胞凋亡。由于死细胞不能产生，即caspase-3/7活性本质上是短暂的;因此分析应该在30分钟至4小时后挑战之间完成，这取决于细胞应激的效果。细胞活力是成反比的胱天蛋白酶3/7活性，并且可以用于确定细胞死亡的机制。例如，该方法以前已经用来表明预处理与肽激素食欲减少细胞凋亡的挑战与过氧化氢 ​​的下丘脑细胞，这表明影响此治疗机制在防止氧化损伤10重要。要注意这些试验是细胞系和组织依赖性的，因为它们依赖于线粒体活性降低测定试剂是非常重要的。该协议已被优化，成年小鼠的下丘脑（A12）细胞;然而，所描述的方法可以被改变，以适应类似研究的范围之内。 从一个单一的复用培养测定以及提供了对个体进行测定几个原因的传统方法的优点。除了 ​​节省时间，细胞样本，并培养试剂，复分析还可以提供细胞存活和死亡的知识，提供内部控制，并省去了重复实验，11，12。 可供选择的方法都依赖于蛋白质印迹或ELISA中，这是可靠的测定法，但价格昂贵和费时的（1-2天），使用96孔形式时尤其如此。不含花费来培养细胞为复测定的时间，总时间不超过3小时。虽然该协议已被优化，与A12单元格的使用，它可以同时考虑的因素可能影响细胞的完整性来改变用于其他车型。阻止如果该协议将工作的一系列实验采矿取决于样本或实验条件的数量以及规划下游实验。

<table border="0" cellpadding="0" cellspacing="0" width="723">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:268px;">Adult Mouse Hypothalamus Cell Line mHypoA-1/2&#160;</td>
			<td style="width:137px;">Cellutions Biosystems Inc.</td>
			<td style="width:151px;">CLU172</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Dulbecco&#8217;s Modified Eagle&#8217;s Medium</td>
			<td style="width:137px;">Invitrogen</td>
			<td style="width:151px;">10313-039</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Fetal bovine serum&#160;</td>
			<td style="width:137px;">PAA Labs</td>
			<td style="width:151px;">A15-751</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Penicillin/Streptomycin</td>
			<td style="width:137px;">Invitrogen</td>
			<td style="width:151px;">15070-063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Palmitic Acid</td>
			<td style="width:137px;">Sigma-Aldrich</td>
			<td style="width:151px;">P0500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Dimethy sulfoxide&#160;</td>
			<td style="width:137px;">Sigma-Aldrich</td>
			<td style="width:151px;">D2650</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:268px;"> 
			PrestoBlue Cell Viability Reagent</td>
			<td style="width:137px;">Invitrogen</td>
			<td>A13262</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Caspase-Glo 3/7 Assay Systems</td>
			<td style="width:137px;">Promega</td>
			<td style="width:151px;">G8091</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Equipment</td>
			<td style="width:137px;"></td>
			<td style="width:151px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:268px;">96W Optical Bottom Plate, Black Polystyrene, Cell Culture Treated, with lid, Sterile</td>
			<td style="width:137px;">Thermo Fisher Scientific</td>
			<td style="width:151px;">165305</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:268px;">SpectraMax M5 Multi-Mode Microplate</td>
			<td style="width:137px;">Molecular Devices</td>
			<td style="width:151px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

use of a caspase multiplexing assay to determine apoptosis in a hypothalamic cell model

复测定法中的复杂的细胞机制的研究的能力，省去了重复的实验中，提供了内部控制，并在成本和试剂减少浪费。这里，我们描述了多重测定，以评估细胞凋亡的优化以下棕榈酸（PA）的挑战， 在体外模型中的下丘脑，同时使用荧光和发光为基础的分析来测量在96孔的活细胞计数和caspase-3/7活性微量滴定板形式。下面的PA挑战，存活细胞用刃天青类荧光测定法测定。胱天蛋白酶3/7活性是再使用一个luminogenic基板，DEVD，并且标准化为细胞数目确定。此复用测定法是用于确定变化caspase活性以下的凋亡刺激物，如饱和脂肪酸挑战的有效技术。饱和脂肪酸的PA可以增加下丘脑的氧化应激和细胞凋亡，表明了潜在importa在研究饱和脂肪酸和神经元功能之间的关系描述此处测定法如的考。

上述协议描述的结果在复用两个独立的实验，以确定细胞死亡的机制。 图1显示了协议的概述，以确定细胞活力和caspase-3/7活性。半胱天冬酶活性的细胞后2小时温育（ 图2A和表1）挑战与PA显著增加。的细胞膜完整性的丧失与细胞凋亡的诱导，这是明显的细胞后2小时暴露到PA（ 图2B）相关的形态学变化， 图3概括了PA诱导细胞凋亡，并演...

Watch this Scientific Journal Video about Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model at JoVE.com

Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model

多重分析可以提供有益的信息为基础的细胞机制，消除试剂和不必要的重复实验的浪费。我们在这里描述的多路胱天蛋白酶3/7活性测定法，使用荧光和发光为基础的方法，以确定在以下与棕榈酸的氧化挑战的体外模型中的下丘脑细胞活力。

使用胱天蛋白酶复分析，以确定细胞凋亡的下丘脑细胞模型

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and ...

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Neuroscience

14.9K 次观看.  Minneapolis Veterans Affairs Health Care System. 多重分析可以提供有益的信息为基础的细胞机制，消除试剂和不必要的重复实验的浪费。我们在这里描述的多路胱天蛋白酶3/7活性测定法，使用荧光和发光为基础的方法，以确定在以下与棕榈酸的氧化挑战的体外模型中的下丘脑细胞活力。 

视频: Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve 
transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous
 chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals 
through the vitreous fluid ensures that they act upon the entire RGC population. Moreover, RGCs can be selectively transfected by applying short interfering RNAs 
(siRNAs), plasmids, or viral vectors to the cut end of the optic nerve 5-7 or injecting vectors into their target, the superior colliculus 8. 
This allows researchers to study apoptotic mechanisms in the desired neuronal population without confounding effects on other bystander neurons or surrounding glia. 
An additional benefit is the ease and accuracy with which cell survival can be quantified after injury. The retina is a flat, layered tissue and RGCs are localized in 
the innermost layer, the ganglion cell layer. The survival of RGCs can be tracked over time by applying a fluorescent tracer (3% Fluorogold) to the cut end of the 
optic nerve at the time of axotomy, or by injecting the tracer into the superior colliculus (RGC target) one week prior to axotomy. The tracer is retrogradely transported, labeling 
the entire RGC population. Because the ganglion cell layer is a monolayer (one cell thick), RGC densities can be quantified in flat-mounted tissue, without the need 
for stereology. Optic nerve transection leads to the apoptotic death of 90% of injured RGCs within 14 days postaxotomy 9-11. RGC apoptosis has a 
characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a time window for 
experimental manipulations directed against pathways involved in apoptosis.

Optic Nerve transection is a widely used model of adult CNS injury. Ninety percent of retinal ganglion cells (RGCs) whose axons are completely transected (axotomy) die within 14 days after axotomy. This model is easily amenable to experimental manipulations and highly reproducible.

视神经横断：一个成人在中枢神经系统的神经元凋亡模型

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be ...

科研

神经科学

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

神经细胞集落形成细胞分析检测，以区分善意的神经祖细胞的神经干细胞

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depolarization

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a reduction in neuronal population and function. Damaged axons can release excitatory molecules including potassium or glutamate into the extracellular matrix, which in turn, can produce further insult and injury to the supporting glial cells including astrocytes and oligodendrocytes 8, 16. If the insult persists, cells will undergo programmed cell death (apoptosis), which is regulated and activated by a number of well-established signal transduction cascades 14. Apoptosis and tissue necrosis can occur after traumatic brain injury, cerebral ischemia, and seizures. A classical example of apoptotic regulation is the family of cysteine-dependent aspartate-directed proteases, or caspases. Activated proteases including caspases have also been implicated in cell death in response to chronic neurodegenerative diseases including Alzheimer's, Huntington's, and Multiple Sclerosis 4, 14, 3, 11, 7.
In this protocol we describe the use of the NucView 488 caspase-3 substrate to measure the rate of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte (OLG) cell cultures 15, 5, following exposure to different extracellular stresses such as high concentrations of potassium or glutamate. The conditionally-immortalized N19-OLG cell line (representing the O2A progenitor) was obtained from Dr. Anthony Campagnoni (UCLA Semel Institute for Neuroscience) 15, 5, and has been previously used to study molecular mechanisms of myelin gene expression and signal transduction leading to OLG differentiation (e.g.6, 10). We have found this cell line to be robust with respect to transfection with exogenous myelin basic protein (MBP) constructs fused to either RFP or GFP (red or green fluorescent protein) 13, 12. Here, the N19-OLG cell cultures were treated with either 80 mM potassium chloride or 100 mM sodium glutamate to mimic axonal leakage into the extracellular matrix to induce apoptosis 9. We used a bi-functional caspase-3 substrate containing a DEVD (Asp-Glu-Val-Asp) caspase-3 recognition subunit and a DNA-binding dye 2. The substrate quickly enters the cytoplasm where it is cleaved by intracellular caspase-3. The dye, NucView 488 is released and enters the cell nucleus where it binds DNA and fluoresces green at 488 nm, signaling apoptosis. Use of the NucView 488 caspase-3 substrate allows for live-cell imaging in real-time 1, 10. In this video, we also describe the culturing and transfection of immortalized N19-OLG cells, as well as live-cell imaging techniques.

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depola

Live-cell imaging of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte cell cultures using the NucView 488 caspase-3 substrate. This technique is applicable for programmed cell death assays in real-time in a variety of cell types and tissues.

监测劈开caspase - 3活性和钾诱导膜去极化使用活细胞成像和Cleaveable荧光染料基板的永生的少突胶质细胞凋亡

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a ...

Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral delivery or transgenic animals have potential confounds that make interpretation of results from these studies difficult. New radiological tools are emerging, however, that allow one to noninvasively investigate the function of select groups of adult-born neurons through accurate and precise anatomical targeting in small animals. Focal ionizing radiation inhibits the birth and differentiation of new neurons, and allows targeting of specific neural progenitor regions. In order to illuminate the potential functional role that adult hypothalamic neurogenesis plays in the regulation of physiological processes, we developed a noninvasive focal irradiation technique to selectively inhibit the birth of adult-born neurons in the hypothalamic median eminence. We describe a method for Computer tomography-guided focal irradiation (CFIR) delivery to enable precise and accurate anatomical targeting in small animals. CFIR uses three-dimensional volumetric image guidance for localization and targeting of the radiation dose, minimizes radiation exposure to nontargeted brain regions, and allows for conformal dose distribution with sharp beam boundaries. This protocol allows one to ask questions regarding the function of adult-born neurons, but also opens areas to questions in areas of radiobiology, tumor biology, and immunology. These radiological tools will facilitate the translation of discoveries at the bench to the bedside.

The function of adult-born mammalian neurons remains an active area of investigation. Ionizing radiation inhibits the birth of new neurons. Using computer tomography-guided focal irradiation (CFIR), three-dimensional anatomical targeting of specific neural progenitor populations can now be used to assess the functional role of adult neurogenesis.

成人下丘脑神经发生局灶性放射性抑制功能审讯

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral ...

A Novel Light Damage Paradigm for Use in Retinal Regeneration Studies in Adult Zebrafish

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, photoreceptor degeneration triggers M&#252;ller glial cells to re-enter the cell cycle and produce transient-amplifying progenitors. These progenitors continue to proliferate as they migrate to the damaged area, where they ultimately give rise to new photoreceptors. Currently, there are two widely-used LIRD paradigms, each of which results in varying degrees of photoreceptor loss and corresponding differences in the regeneration response. As more genetic and pharmacological tools are available to test the role of individual genes of interest during regeneration, there is a need to develop a robust LIRD paradigm. Here we describe a LIRD protocol that results in widespread and consistent loss of both rod and cone photoreceptors in which we have combined the use of two previously established LIRD techniques. Furthermore, this protocol can be extended for use in pigmented animals, which eliminates the need to maintain transgenic lines of interest on the albino background for LIRD studies.

Multiple light damage protocols have been described to damage photoreceptors and consequently induce a retinal regeneration response in adult zebrafish. This protocol describes an improved method that can be used in pigmented animals and that damages the vast majority of rod and cone photoreceptors across the entire retina.

成年斑马鱼视网膜再生研究中使用的一种新型的光损伤范式

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, ...

Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay described here will aid in elucidating the role of microglia in neuroinflammation and neurodegenerative diseases. The use of microglia, resident immune cells of the brain, as a surrogate biosensor provides novel insight into how inflammatory responses mediate neuronal insults. Here, we utilize an immortalized murine microglial cell line, designated BV2, and describe a method for nanoparticle exposure using silver nanoparticles (AgNPs) as a standard. We describe how to expose microglia to nanoparticles, how to remove nanoparticles from supernatant, and how to use supernatant from activated microglia to determine toxicity, using hypothalamic cell survival as a measure. Following AgNP exposure, BV2 microglial activation was validated using a tumor necrosis factor alpha (TNF-&#945;) enzyme linked immunosorbent assay (ELISA). The supernatant was filtered to remove the AgNP and to allow cytokines and other secreted factors to remain in the conditioned media. Hypothalamic cells were then exposed to supernatant from AgNP activated microglia and survival of neurons was determined using a resazurin-based fluorescent assay. This technique is useful for utilizing microglia as a surrogate biomarker of neuroinflammation and determining the effect of neuroinflammation on other cell types.

Microglia (immune cells of the brain), are used as a surrogate biosensor to determine how nanoparticles influence neurotoxicity. We describe a series of experiments designed to assay microglial response to nanoparticles and exposure of hypothalamic neurons to supernatant from activated microglia to determine neurotoxicity.

小胶质细胞作为替代生物传感器确定纳米颗粒神经毒性

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

使用的跟踪经典条件反射，评估胎儿酒精谱系障碍大鼠模型海马功能障碍

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. To efficiently and accurately deorphanize ORs, we combine the use of a heterologous cell line to express mammalian ORs and a genetically modified biosensor plasmid to measure cAMP production downstream of OR activation in real time. This assay can be used to screen odorants against ORs and vice versa. Positive odorant-receptor interactions from the screens can be subsequently confirmed by testing against various odor concentrations, generating concentration-response curves. Here we used this method to perform a high-throughput screening of an odorous compound against a human OR library expressed in Hana3A cells and confirmed that the positively-responding receptor is the cognate receptor for the compound of interest. We found this high-throughput detection method to be efficient and reliable in assessing OR activation and our data provide an example of its potential use in OR functional studies.

Characterizing the function of odorant receptors serves an indispensable part in the deorphanization process. We describe a method to measure the activation of odorant receptors in real time using a cAMP assay.

实时 cAMP 检测气味受体激活的活细胞测量

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. ...

A Protocol for Measuring Cue Reactivity in a Rat Model of Cocaine Use Disorder

Cocaine use disorder (CUD) follows a trajectory of repetitive self-administration during which previously neutral stimuli gain incentive value. Cue reactivity, the sensitivity to cues previously linked with the drug-taking experience, plays a prominent role in human craving during abstinence. Cue reactivity can be assessed as the attentional orientation toward drug-associated cues that is measurable as appetitive approach behavior in both preclinical and human studies. Herein describes an assessment of cue reactivity in rats trained to self-administer cocaine. Cocaine self-administration is paired with the presentation of discrete cues that act as conditioned reinforcers (i.e., house light, stimulus light, infusion pump sounds). Following a period of abstinence, lever presses in the cocaine self-administration context accompanied by the discrete cues previously paired with cocaine infusion are measured as cue reactivity. This model is useful to explore neurobiological mechanisms underlying cue reactivity processes as well as to assess pharmacotherapies to suppress cue reactivity and therefore, modify relapse vulnerability. Advantages of the model include its translational relevance, and its face and predictive validities.&#160;The primary limitation of the model is that the cue reactivity task can only be performed infrequently and must only be used in short duration (e.g., 1 hour), otherwise rats will begin to extinguish the pairing of the discrete cues with the cocaine stimulus. The model is extendable to any positively reinforcing stimulus paired with discrete cues; though particularly applicable to drugs of abuse, this model may hold future applications in fields such as obesity, where palatable food rewards can act as positively reinforcing stimuli.

Cue reactivity is conceptualized as sensitivity to cues linked with drug-taking experiences that contribute to craving and relapse in abstinent humans. Cue reactivity is modeled in rats by measuring attentional orientation toward drug-associated cues that results in appetitive approach behavior in a cue reactivity test following self-administration and forced abstinence.

可卡因使用障碍大鼠模型中提示反应性测定的一种协议

Cocaine use disorder (CUD) follows a trajectory of repetitive self-administration during which previously neutral stimuli gain incentive value. Cue ...

A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied in vitro using the neurosphere assay. This colony-forming assay is a powerful tool to study the response of NSCs to exogenous factors in a dish; however, this can also be used to study the effect of in vivo manipulations with the proper understanding of the strengths and limitations of the assay. One manipulation of the clinical interest is the effect of injury on endogenous NSC activation. Current models of spinal cord injury provide a challenge to study this as the severity of common contusion, compression, and transection models cause the destruction of the NSC niche at the site of the injury where the stem cells reside. Here, we describe a minimal injury model that causes localized damage at the superficial dorsolateral surface of the lower thoracic level (T7/8) of the adult mouse spinal cord. This injury model spares the central canal at the level of injury and permits analysis of the NSCs that reside at the level of the lesion at various time points following injury. Here, we show how the neurosphere assay can be utilized to study the activation of the two distinct, lineally-related, populations of NSCs that reside in the spinal cord periventricular region - primitive and definitive NSCs (pNSCs and dNSCs, respectively). We demonstrate how to isolate and culture these NSCs from the periventricular region at the level of injury and the white matter injury site. Our post-surgical spinal cord dissections show increased numbers of pNSC and dNSC-derived neurospheres from the periventricular region of injured cords compared to controls, speaking to their activation via injury. Furthermore, following injury, dNSC-derived neurospheres can be isolated from the injury site &#8212; demonstrating the ability of NSCs to migrate from their periventricular niche to sites of injury.

Here, we demonstrate the performance of a minimal spinal cord injury model in an adult mouse that spares the central canal niche housing endogenous neural stem cells (NSCs). We show how the neurosphere assay can be used to quantify activation and migration of definitive and primitive NSCs following injury.

小鼠脊髓损伤模型中内源性神经干细胞活化的干细胞检测

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied ...