我们感谢华盛顿大学的雷蒙德·桑切斯在手稿编辑和 M.Sc 方面给予的宝贵帮助。 乔治娜·科尔特斯和辛蒂亚·哈维尔 M.Sc 在这项技术标准化方面提供了技术支持。我们也感谢萨拉戈萨省高级兽医服务机构的成员：MVZ。阿德里亚娜·阿尔塔米拉诺，MVZ罗曼·埃尔南德斯和MVZ多洛雷斯-伊丽莎白·古斯芒为实验动物提供出色的维护和护理。本协议中描述的实验得到了DGAPA-PAPIIT赠款编号：IN216015和CONACyT赠款编号的支持：236908罗伯托·多明格斯。卡洛斯-卡米洛·席尔瓦是墨西哥国立大学生物学博士项目的博士生，并得到墨西哥国家科学与技术委员会（赠款编号：294555）的支持。

作者对本文没有什么可透露的。

本文描述了一种在任何给定时间暂时灭活清醒和无拘无束的老鼠大脑中一个离散区域的方法。还提供了一个简单的方法来跟踪他们的排卵周期和评估排卵。此协议允许通过比较TTX处理动物的排卵结果与车辆处理动物的排卵结果，直接分析特定大脑区域对驱动排卵机制的贡献。除了神经科学实验室常见的立体定向仪器和微注射泵外，这种方法不需要昂贵的材料。商用罐体和微喷射器是这类实验的常?...

排卵是每个卵巢/月经周期从卵巢中释放一个或多个成熟卵母细胞的过程。由于所有哺乳动物物种都依赖于游戏品种的生产，对调节排卵机制的理解在生物医学、畜牧业和濒危物种的维持等领域产生了巨大影响。排卵由下丘脑-垂体-卵巢轴调节，该轴涉及几个下丘脑和下垂外区域、前垂体中的角膜和肉芽细胞，这些细胞与卵母细胞一起形成卵巢内的卵巢卵泡1。
卵巢卵泡生长，发展，并最终排卵，以回应卵泡刺激激素和叶黄素化激素的补品和阶段性分泌，由卵泡激素分泌的两个卵巢激素。戈纳多特罗平分泌模式是适当的卵泡发育和排卵的关键，它是由戈纳多特罗平释放激素 （GnRH）1，2调节。这种神经肽由分散在基底脑中的神经元合成，然后分泌到连接下丘脑和前垂体的门户血管。GnRH神经元的分泌活动反过来又由不同大脑结构产生的突触输入调节。这些结构传达了有关生物体外部和内部环境状况的信息，包括食物的供应、光佩里德的长度和血液中激素的浓度。从这个意义上说，它们塑造了每个物种的生殖模式，必须确定这些结构的具体作用，以便正确理解排卵的机制。例如，已经表明，在最恶劣的周期中，雌二醇水平的波动调节了GnRH的分泌：然而，GnRH神经元并不表示检测此类变化所需的雌二醇受体等形。表达这些受体的两个神经元群分别位于第三心室的椎间垂直区域和弧形细胞核中，并且与GnRH神经元有稳定突触。有证据表明，这些神经元解释雌二醇的浓度，然后刺激GnRH神经元的活性，释放吻肽，GnRH分泌物3的有力诱导剂。
涉及热病变或化学病变以及机械耳聋的实验使研究人员能够确定几个大脑结构参与排卵4、5、6、7、8、9、10、11、12的调节.然而，这些实验具有侵入性和创伤性的缺点，在评估治疗效果之前需要几天的恢复时间，从而妨碍了对治疗急性效应的分析。此外，它们长期影响目标区域并中断其他生理过程。由于这些问题，这些实验的结果通常被动物体内的内在补偿机制所掩盖，并提取有关所涉及区域的时间调节动态的准确信息是相当困难的。
通过引导性大麻酶暂时破坏神经元活动的药物的微注射是一种适当的替代方法，可以超越上述缺点。通过立体定向手术，可以将金核酸放入任何大脑区域，使研究人员能够在手术的混淆效应消失后开始药物治疗。药物的定时微注射使研究人员能够测试有关该地区对过程特定步骤的贡献的假设，并且可以在清醒的受约束或自由移动的动物中执行。各种药物，包括局部麻醉剂、激动剂、拮抗剂、反激动剂和生物毒素，如四毒素（TTX），可在特定时间微注射到感兴趣的区域。
TTX是一种生物毒素，由生活在河豚体内的细菌以及其他脊椎动物和无脊椎动物合成。TTX通过对钠通道的选择性和瞬态封锁来抑制神经活动，从而抑制依赖钠的行为潜力。在TTX的存在下，细胞在去极化阶段经历改变，因此不能激发，但仍然活着。TTX的阻塞效应是由其分子组成解释的：一个硅基组能够通过钠通道的细胞外方面，但分子的其余部分由于其大小而无法通过，因此它被卡住并阻塞了通道13、14、15、16、17.TTX 的作用机制允许它作为研究体外和体内神经系统的工具。这种毒素的内脑注射已用于研究离散脑区在几个过程中的作用，如记忆保留18，睡眠和觉醒19，位置识别20，空间导航21，药物滥用22，热调节23，精神分裂症24的发展，性行为25和排卵调节26 除其他外。在这个协议中，我们描述了在清醒和无拘无束的大鼠中TTX微注射对下丘脑核短暂灭活排卵的影响。

<table>
	<tbody>
		<tr>
			<td>10 &#956;L Hamilton syringes</td>
			<td>Hamilton</td>
			<td>80314</td>
			<td></td>
		</tr>
		<tr>
			<td>21 G x 1&#34; stainless steel hypdermic needle</td>
			<td>BD</td>
			<td>305165</td>
			<td></td>
		</tr>
		<tr>
			<td>23 G x 1&#34; stainless steel hypdermic needle</td>
			<td>BD</td>
			<td>305145</td>
			<td></td>
		</tr>
		<tr>
			<td>30 G x 1/2&#34; stainless steel hypdermic needle</td>
			<td>BD</td>
			<td>305106</td>
			<td></td>
		</tr>
		<tr>
			<td>Artificial cerebrospinal fluid</td>
			<td>BASi</td>
			<td>MD-2400</td>
			<td></td>
		</tr>
		<tr>
			<td>Bone trimer</td>
			<td>Fine Science Tools</td>
			<td>16152-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Burr for micro drill</td>
			<td>Fine Science Tools</td>
			<td>19007-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Clipper</td>
			<td>Wahl</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cut-off disc</td>
			<td>Dremel</td>
			<td>SM5010</td>
			<td></td>
		</tr>
		<tr>
			<td>Cutting tweezers</td>
			<td>Truper</td>
			<td>17367</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanocrylate glue</td>
			<td>Kola loka</td>
			<td>K-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Dental cement</td>
			<td>Nic Tone</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Enrofloxasin</td>
			<td>Senosiain</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin</td>
			<td>Sigma</td>
			<td>E4009</td>
			<td></td>
		</tr>
		<tr>
			<td>Estereoscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Extra fine Bonn scissors</td>
			<td>Fine Science Tools</td>
			<td>14084-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Face mask</td>
			<td>Lanceta HG</td>
			<td>60036</td>
			<td></td>
		</tr>
		<tr>
			<td>Graefe Forceps</td>
			<td>Fine Science Tools</td>
			<td>11050-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxilin</td>
			<td>Sigma</td>
			<td>H3136</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemostats</td>
			<td>Fine Science Tools</td>
			<td>13008-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot bead sterilizer</td>
			<td>Fine Science Tools</td>
			<td>18000-45</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Sigma</td>
			<td>320331</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypromelose artificial tears</td>
			<td>Sophia Labs</td>
			<td>8950015</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Pisa Agropecuaria</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Meloxicam</td>
			<td>Aranda</td>
			<td>1183</td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjection pump</td>
			<td>KD Scientific</td>
			<td>788380</td>
			<td></td>
		</tr>
		<tr>
			<td>Monomer</td>
			<td>Nic Tone</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mototool</td>
			<td>Dremel</td>
			<td>3000</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrile gloves</td>
			<td>Lanceta HG</td>
			<td>69028</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-Rupture Ear Bars</td>
			<td>David Kopf Instruments</td>
			<td>855</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L lysine</td>
			<td>Sigma</td>
			<td>P4707</td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone-iodine</td>
			<td>Dermo Dine</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone-iodine with soap</td>
			<td>Germisin espuma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure tweezers</td>
			<td>Truper</td>
			<td>17371</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anesthesia mask</td>
			<td>David Kopf Instruments</td>
			<td>Model 906</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline solution</td>
			<td>PISA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Fine Science Tools</td>
			<td>10004-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blade</td>
			<td>Fine Science Tools</td>
			<td>10015-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pentobarbital</td>
			<td>Pisa Agropecuaria</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Standard electrode holder</td>
			<td>David Kopf Instruments</td>
			<td>1770</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel wire</td>
			<td>American Orthodontic</td>
			<td>856-612</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotaxic apparatus</td>
			<td>David Kopf Instruments</td>
			<td>Model 900LS</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Sissors</td>
			<td>Fine Science Tools</td>
			<td>14001-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Teflon connectors</td>
			<td>Basi</td>
			<td>MD-1510</td>
			<td></td>
		</tr>
		<tr>
			<td>Teflon tubing</td>
			<td>Basi</td>
			<td>MF-5164</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrodotoxin</td>
			<td>Alomone labs</td>
			<td>T-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Vaporizer</td>
			<td>Kent scientific</td>
			<td>VetFlo</td>
			<td></td>
		</tr>
	</tbody>
</table>

unraveling the role of discrete areas of the rat brain in the regulation of ovulation through reversible inactivation by tetrodotoxin microinjections

许多实验方法被用于研究大脑在排卵调节中的作用。例子包括神经元组的病变和耳聋，这两种侵入性方法都永久地损害了目标区域的完整性。这些方法还附带附带影响，可影响对急性和时效监管机制的分析。针对特定大脑区域的导引罐体的立体植入，然后是恢复期，使研究人员能够在手术的不良效果消失后微观注射不同的药物。四毒素已被用于确定几个大脑区域在各种生理过程中的作用，因为它暂时抑制了依赖钠的作用潜力，从而阻断了目标区域的所有神经活动。本协议将此方法与评估卵巢周期和排卵的策略相结合，以揭示离散的大脑区域在排卵调节中在最恶劣周期的任何特定阶段的作用。觉醒和无拘无束的老鼠（Rattus norvegicus）被用来避免麻醉剂和应激激素对排卵的阻塞作用。此协议可以很容易地适应其他物种，大脑目标和药理制剂，以研究不同的生理过程。该方法的未来改进包括使用小直径的玻璃毛细血管而不是导引罐体设计微型喷射系统。这将减少植入过程中受损的组织数量，并减少注入药物在目标区域外的扩散。

上述协议通过评估单个TTX或车辆（人工脑脊液）的影响进行了测试：ACSF）微注射成两个不同的细胞核之一，已知参与调节排卵在老鼠：超囊性原子核和弧形核。之所以选择超乳酸核，是因为它含有哺乳动物的中央昼夜起搏器。它参与循环事件的监管，作为戈纳多特罗平的分泌物。之所以选择弧形细胞核，是因为它包含大量表达雌二醇受体的神经元，在大多数绝经周期中刺激GnRH分泌。微注射是在14...

Watch this Scientific Journal Video about Unraveling the Role of Discrete Areas of the Rat Brain in the Regulation of Ovulation through Reversible Inactivation by Tetrodotoxin Microinjections at JoVE.

Unraveling the Role of Discrete Areas of the Rat Brain in the Regulation of Ovulation through Reversible Inactivation by Tetrodotoxin Microinjections

本协议描述了低成本微注射系统的构建、对深脑结构的立体定向植入以及清醒和无拘无束的大鼠对四毒素定时微注射的程序。目标是通过抑制下丘脑结构的神经活动来揭示下丘脑结构参与排卵的调节。

通过特洛多毒素微注射可逆灭活，揭开鼠脑离散区在排卵调节中的作用

Many experimental approaches have been used for studying the role of the brain in the regulation of ovulation. Examples include the lesion and ...

This protocol allows researchers to explore the nuclei of the neuronal circuits that regulates ovulation in female mammals at specific times of their estrous cycle.This technique can be adapted to study any process on the regulation of the brain in any species.Also they can less art fabricator in the lab lowering the overall cost.Demonstrating this procedure will be Montserrat Bola

unraveling-role-discrete-areas-rat-brain-regulation-ovulation-through

Research

JoVE Journal

Neuroscience

3.0K 次观看.  Universidad Nacional Autónoma de México. This protocol allows researchers to explore the nuclei of the neuronal circuits that regulates ovulation in female mammals at specific times of their estrous cycle.This technique can be adapted to study any process on the regulation of the brain in any species.Also they can less art fabricator in the lab lowering the overall cost.Demonstrating this procedure will be Montserrat Bola

视频: Unraveling the Role of Discrete Areas of the Rat Brain in the Regulation of Ovulation through Reversible Inactivation by Tetrodotoxin Microinjections

Brain Imaging Investigation of the Neural Correlates of Emotion Regulation

The ability to control/regulate emotions is an important coping mechanism in the face of emotionally stressful situations. Although significant progress has been made in understanding conscious/deliberate emotion regulation (ER), less is known about non-conscious/automatic ER and the associated neural correlates. This is in part due to the problems inherent in the unitary concepts of automatic and conscious processing1. Here, we present a protocol that allows investigation of the neural correlates of both deliberate and automatic ER using functional magnetic resonance imaging (fMRI). This protocol allows new avenues of inquiry into various aspects of ER. For instance, the experimental design allows manipulation of the goal to regulate emotion (conscious vs. non-conscious), as well as the intensity of the emotional challenge (high vs. low). Moreover, it allows investigation of both immediate (emotion perception) and long-term effects (emotional memory) of ER strategies on emotion processing. Therefore, this protocol may contribute to better understanding of the neural mechanisms of emotion regulation in healthy behaviour, and to gaining insight into possible causes of deficits in depression and anxiety disorders in which emotion dysregulation is often among the core debilitating features.

We present a protocol that allows investigation of the neural correlates of deliberate and automatic emotion regulation, using functional magnetic resonance imaging. This protocol can be used in healthy participants, both young and older, as well as in clinical patients.

情绪调节的神经关联的脑成像研究

The ability to control/regulate emotions is an important coping mechanism in the face of emotionally stressful situations. Although significant progress ...

科研

神经科学

Optimized Analysis of DNA Methylation and Gene Expression from Small, Anatomically-defined Areas of the Brain

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to the display of physiological and behavioural phenotypes. Such environmental programming is likely to increase the susceptibility to metabolic, cardiovascular and mental diseases 3,4.
DNA methylation and histone modifications are considered key processes in the mediation of the gene-environment dialogue and appear also to underlay environmental programming 5. In mammals, DNA methylation typically comprises the covalent addition of a methyl group at the 5-position of cytosine within the context of CpG dinucleotides.
CpG methylation occurs in a highly tissue- and cell-specific manner making it a challenge to study discrete, small regions of the brain where cellular heterogeneity is high and tissue quantity limited. Moreover, because gene expression and methylation are closely linked events, increased value can be gained by comparing both parameters in the same sample.
Here, a step-by-step protocol (Figure 1) for the investigation of epigenetic programming in the brain is presented using the 'maternal separation' paradigm of early life adversity for illustrative purposes. The protocol describes the preparation of micropunches from differentially-aged mouse brains from which DNA and RNA can be simultaneously isolated, thus allowing DNA methylation and gene expression analyses in the same sample.

A streamlined workflow to study DNA methylation and gene expression changes upon early-life stress is shown. Starting from maternal separation of newborn mice and isolation of discrete brain tissues, we represent a protocol to simultaneously isolate DNA and RNA from brain tissue punches for subsequent bisulfite sequencing and RT-PCR analysis.

优化小，解剖定义的大脑区域的DNA甲基化和基因表达分析

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to ...

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the function of various brain regions for behavior or other experimental outcomes is to implement a localized ablation of function. In humans, patient populations with localized brain lesions are often studied for deficits, in hopes of revealing the underlying function of the damaged area. In rodents, one can experimentally induce lesions of specific brain regions.
Lesion can be accomplished in several ways. Electrolytic lesions can cause localized damage but will damage a variety of cell types as well as traversing fibers from other brain regions that happen to be near the lesion site. Inducible genetic techniques using cell-type specific promoters may also enable site-specific targeting. These techniques are complex and not always practical depending on the target brain area. Excitotoxic lesion using stereotaxic surgery, by contrast, is one of the most reliable and practical methods of lesioning excitatory neurons without damaging local glial cells or traversing fibers.
Here, we present a protocol for stereotaxic infusion of the excitotoxin, N-methyl-D-aspartate (NMDA), into the basolateral amygdala complex. Using anatomical indications, we apply stereotaxic coordinates to determine the location of our target brain region and lower an injection needle in place just above the target. We then infuse our excitotoxin into the brain, resulting in excitotoxic death of nearby neurons. While our experimental subject of choice is a rat, the same methods can be applied to other mammals, with the appropriate adjustments in equipment and coordinates.
This method can be used on a variety of brain regions, including the basolateral amygdala1-6, other amygdala nuclei6, 7, hippocampus8, entorhinal cortex9 and prefrontal cortex10. It can also be used to infuse biological compounds such as viral vectors1, 11. The basic stereotaxic technique could also be adapted for implantation of more permanent osmotic pumps, allowing more prolonged exposure to a compound of interest.

Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

特定脑区的兴奋毒性损伤的成年大鼠立体定向手术治疗

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective treatment for several movement disorders that have failed to respond to medication. Recent progress in the field of DBS surgery has begun to extend the application of this surgical technique to other conditions as diverse as morbid obesity, depression and obsessive compulsive disorder. Despite these expanding indications, little is known about the underlying physiological mechanisms that facilitate the beneficial effects of DBS surgery. One approach to this question is to perform gene expression analysis in neurons that receive the electrical stimulation. Previous studies have shown that neurogenesis in the rat dentate gyrus is elicited in DBS targeting of the anterior nucleus of the thalamus1. DBS surgery targeting the ATN is used widely for treatment refractory epilepsy. It is thus of much interest for us to explore the transcriptional changes induced by electrically stimulating the ATN. In this manuscript, we describe our methodologies for stereotactically-guided DBS surgery targeting the ATN in adult male Wistar rats. We also discuss the subsequent steps for tissue dissection, RNA isolation, cDNA preparation and quantitative RT-PCR for measuring gene expression changes. This method could be applied and modified for stimulating the basal ganglia and other regions of the brain commonly clinically targeted. The gene expression study described here assumes a candidate target gene approach for discovering molecular players that could be directing the mechanism for DBS.

The mechanism underlying the therapeutic effects of Deep Brain Stimulation (DBS) surgery needs investigation. The methods presented in this manuscript describe an experimental approach to examine the cellular events triggered by DBS by analyzing the gene expression profile of candidate genes that can facilitate neurogenesis post DBS surgery.

基因表达的变化大鼠海马后，前丘脑核脑深部刺激分析

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

采用组合运用各种方法，定义质膜交付在轴突分支和神经元形态发生中的作用

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

体内基因转移到雪旺细胞在啮齿动物坐骨神经电穿孔

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

Sublimation of DAN Matrix for the Detection and Visualization of Gangliosides in Rat Brain Tissue for MALDI Imaging Mass Spectrometry

Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass Spectrometry (IMS) experiments. Determining the appropriate protocol to follow throughout the sample preparation process can be difficult as each step must be optimized to comply with the unique characteristics of the analytes of interest. This process involves not only finding a compatible matrix that can desorb and ionize the molecules of interest efficiently, but also selecting the appropriate matrix deposition technique. For example, a wet matrix deposition technique, which entails dissolving a matrix in solvent, is superior for desorption of most proteins and peptides, whereas dry matrix deposition techniques are particularly effective for ionization of lipids. Sublimation has been reported as a highly efficient method of dry matrix deposition for the detection of lipids in tissue by MALDI IMS due to the homogeneity of matrix crystal deposition and minimal analyte delocalization as compared to many wet deposition methods 1,2. Broadly, it involves placing a sample and powdered matrix in a vacuum-sealed chamber with the samples pressed against a cold surface. The apparatus is then lowered into a heated bath (sand or oil), resulting in sublimation of the powdered matrix onto the cooled tissue sample surface. Here we describe a sublimation protocol using 1,5-diaminonaphthalene (DAN) matrix for the detection and visualization of gangliosides in the rat brain using MALDI IMS.

A protocol for the sublimation of DAN matrix onto rat brain tissue for the detection of gangliosides using MALDI Imaging Mass Spectrometry is presented.

DAN矩阵升华为神经节苷脂对大鼠脑组织的检测和可视化MALDI成像质谱

Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass ...

Personalized Needles for Microinjections in the Rodent Brain

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to deliver gene or cell therapy products. Unfortunately, current microinjection techniques use steel or glass needles that are suboptimal for multiple reasons: in particular, steel needles may cause tissue damage, and glass needles may bend when lowered deeply into the brain, missing the target region. In this article, we describe a protocol to prepare and use quartz needles that combine a number of useful features. These needles do not produce detectable tissue damage and, being very rigid, ensure reliable delivery in the desired brain region even when using deep coordinates. Moreover, it is possible to personalize the design of the needle by making multiple holes of the desired diameter. Multiple holes facilitate the injection of large amounts of solution within a larger area, whereas large holes facilitate the injection of cells. In addition, these quartz needles can be cleaned and re-used, such that the procedure becomes cost-effective.

We describe here a protocol for microinjection in the rodent brain that uses quartz needles. These needles do not produce detectable tissue damage and ensure reliable delivery even in deep regions. Moreover, they can be adapted to research needs by personalized designs and can be re-used.

啮齿动物脑 Microinjections 的个性化针

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

小容积回收-灌注-浸没式室系统中维持的急性海马切片的突触可塑性记录

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Translational Brain Mapping at the University of Rochester Medical Center: Preserving the Mind Through Personalized Brain Mapping

The Translational Brain Mapping Program at the University of Rochester is an interdisciplinary effort that integrates cognitive science, neurophysiology, neuroanesthesia, and neurosurgery. Patients who have tumors or epileptogenic tissue in eloquent brain areas are studied preoperatively with functional and structural MRI, and intraoperatively with direct electrical stimulation mapping. Post-operative neural and cognitive outcome measures fuel basic science studies about the factors that mediate good versus poor outcome after surgery, and how brain mapping can be further optimized to ensure the best outcome for future patients. In this article, we describe the interdisciplinary workflow that allows our team to meet the synergistic goals of optimizing patient outcome and advancing scientific understanding of the human brain.

This article provides an overview of a multi-modal brain mapping program designed to identify regions of the brain that support critical cognitive functions in individual neurosurgery patients.

罗切斯特大学医学中心的转化脑图:通过个性化大脑映射保护大脑

The Translational Brain Mapping Program at the University of Rochester is an interdisciplinary effort that integrates cognitive science, neurophysiology, ...