作者感谢彼得Gerrits博士在视频中使用的图纸。

注:防腐技术，应该在整个过程中就业。手术过程中，将使用所有的工具和材料（棉拭子，纱布等），应通过高压灭菌消毒。应戴口罩，头发的帽子和无菌手套。工作区和立体定位仪上，应彻底清洗，并用70％乙醇溶液消毒。 1。手术前程序<ol><li>设立立体定位仪和所需的所有材料。预温加热垫。 </li><li>将在其支持的套管，并检查它是否是直线。 </li><li>打开气系统 - 环境空气和氧气（总流量的30-35％是氧气）的混合物。 </li><li>称取大鼠和管理的麻醉。我们使用的是皮下注射氯胺酮（37.5毫克/千克）和右旋美托咪啶（0.25毫克/千克）的混合物。不同麻醉协议，看到Flecknell 4和HellebrekerS 等人。7。 </li><li>大鼠后失去了知觉，刮胡子的头部区域，从耳朵之间只是在一个电动剃须刀的眼睛。 </li><li>将加热垫的大鼠，其在气管前面的鼻子。使用血氧饱和度，以确保对大鼠有足够的血氧水平（不应该下降&lt;90％）。请按照正确使用设备制造商的指示。 </li><li>眼霜（Duratears Z，爱尔康）申请在两个眼角膜，以避免脱水。 </li><li>检查老鼠的反射（尾反射或脚趾捏反射，Walantus 等8所示），以确保它有足够的麻醉。如果大鼠继续显示出强劲的反射，可能需要补充麻醉。 </li><li>如果没有脚趾捏反射显示，大鼠放置在立体定位仪，调整耳酒吧，所以，它显示了双方平等的阅读，再次发生空气管在前面的动物固定用鼻子栏。再次检查，如果老鼠显示血氧水平的90％或更高。如果没有，油管调整，使其更接近鼻子，或增加氧气流量。监测血氧水平在整个手术和心脏率。 </li><li>连续监测大鼠直肠温度计（优先连接到一个加热垫）的温度，并记录手术开始和结束的值。调整加热垫或使用毛毯保持体温为37.5到38.5 ° C。 </li></ol> 2。外科<ol><li>注入的镇痛。我们使用的是一个单一卡洛芬（4.0-5.0毫克/公斤，皮下注射）围手术期管理。对于不同的镇痛协议，看到Hellebrekers等人 7 。 </li><li>清洁剃光面积的T他从中央到三次用消毒剂（如洗必泰0.5％）和本地注入利多卡因（20毫克/毫升）和肾上腺素（5毫克/毫升）的混合物，用于局部麻醉和血管收缩（防止头发外围的皮肤出血过多）。 </li><li>作出约2.5厘米的头皮中线前后的切口，直到耳朵后面的眼睛之间。使用4-6猛犬夹捏皮肤，保持切口开放。取出压舌板和/或棉签和清洁的面积暴露颅骨表面有任何结膜组织。 </li><li>检查头部水平:第一，找到的Lambda和引导套管的地方正好在这个位置，触及颅骨。记录背腹的坐标。下一步，将完全超过前囟引导插管，感人的头骨，并记录其背腹坐标。这两个坐标应该是相同的的。如果差值大于0.3毫米，调整鼻栏加以纠正。 </li><li>中号AKE两个小孔用于固定颅骨螺钉，使用消毒手钻（约5毫米的前一个半球的套管位置和其他5毫米，后在对侧半球的套管位置）。放置这些孔的两成无菌螺丝，直到他们没有被完全插入到头骨，紧紧地锚。 </li><li>随着引导导管置于前囟完全，记录前后和横向坐标。添加或减去从囟一个立体图集9-11援助，可以计算出每个大脑区域指导导管放置正确的位置。 </li><li>位置在其正确的位置引导套管，略微触及颅骨。记录背腹的坐标。对于双边插管的位置，找到未来在另一个半球的套管位置，并再次背腹坐标。两个坐标应该是相同的的（或不同&lt;0.3毫米）。 </li><li>用无菌铅笔标记头骨上的套管位置，手钻，颅骨钻孔，检查指导导管援助的规模和正确的位置。一旦孔，使用消毒针头轻轻一拳脑膜允许通畅的导管插入。 </li><li>第一洞放入套管和仔细降低，直到达到最终的腹协调。准备牙科水泥和慷慨申请周围的套管和一个或两个螺丝以固定的套管。等待，直到完全干燥的水泥。之后，小心地取出的背腹杆向上转动套管支持。 </li><li>放置到支持第二套管和套管在另一个半球的位置去。套管成孔，并重复上一步。盖上的螺丝和一个大型水面的套管与水泥，水泥之前是干的，从皮肤移除任何剩余。 </li><li>注入温水（〜37℃）无菌生理盐水（〜10毫升/公斤，SC），以确保补液。 </li><li>水泥已经完全干涸后，取出套管支持，并放入无菌针，每一个套管，以防止阻塞。 </li><li>用无菌生理盐水清洗伤口面积，正面和背部的伤口缝合。 </li><li>从立体定位仪上删除的动物，在它的鼻子前更换气管。继续监测血氧饱和度水平和体温。 </li><li>如果与右美托咪啶注射麻醉剂，注入其拮抗剂atipamezole（0.25毫克/千克，SC），并等待，直到动物唤醒（约5分钟）。 </li><li>大鼠放入一个回收笼。为了避免低温，笼放置在孵化器在28 ° C或加热垫在一个地方，你可以观察到至​​少有一个小时的动物，在返回之前vivarium室。 </li></ol> 3。手术后的护理<ol><li>在<html>手术后第4天，监测老鼠的复苏，保持每日记录的重量和其他有关的动物在实验室记录簿或"动物福利日记"条件的意见。 </li><li>动物疾病，感染的伤口，体重损失或减少福祉必须接受特殊照顾的其他迹象显示明显的迹象:如额外的止痛药剂量，以尽量减少手术后的疼痛，粉的食物和水的混合物除了标准州城刺激老鼠的食欲，和/或皮下注射生理盐水支持补液。 </li><li>如果老鼠没有显示出任何改善这些干预​​措施后，或身体消瘦是&gt; 15％（比手术前体重），牺牲动物麻醉剂过量（人道终点）。 </li><li>大鼠通常需要至少7天的行为实验开始前恢复。 </li></ol> 4。代表性的成果jove_content"&gt;确定是否的各种修改我们的手术过程，特别是氧气补充和连续监测血液氧合水平，心率和体温，，增强动物的生存和提高，手术后其一般条件下，我们比较了非生存率高达20动物（包括20只大鼠）接受手术治疗后，我们实施了这些修改与非生存率与标准协议经营的24个同伙（各20只大鼠）。同伙如图1a所示，非生存率显着降低，与修改后的协议（P &lt;0.05，采用Mann - Whitney U检验，两尾）经营的同伙的样本。此外，由于在图1b显示，减肥手术后手术组大鼠与修改后的协议也显著减少相比，大鼠，操作的标准程序（手术后第1天:P &lt;0.05手术后第2天:P &lt;0.01;手术后第3天:P = 0.17;学生t -检验） <img alt="图1" src="/files/ftp_upload/3528/3528fig1.jpg" /> 图1非成活率和手术后的减肥手术的修改的影响。 （A）非生存率手术组大鼠与修改后的协议作为标准协议与手术组大鼠相比。非存活率（中位数±四分位范围）大鼠的百分比计算，每20只大鼠的队列，没有生存的手术。 * P &lt;0.05，采用Mann - Whitney U检验两个尾（N = 20修改后的协议和标准协议24同伙同伙） 。 （B）减肥（POD1）第一，第二（POD2）和第三（POD3）手术后一天期间（平均± SEM）作为手术前重量的百分比。 * P &lt;0.05，** P &lt;0.01，学生t检验（N = 60）每组。

<ol>
	<li>Russell, W. M. S., Burch, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Principles of Humane Experimental Technique. , (1959).</li><li>Richardson, C. A., Flecknell, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaesthesia+and+post-operative+analgesia+following+experimental+surgery+in+laboratory+rodents:+are+we+making+progress?.">Anaesthesia and post-operative analgesia following experimental surgery in laboratory rodents: are we making progress?.</a> Altern. Lab Anim. 33, 119-127 (2005).</li><li>Stokes, E. L., Flecknell, P. A., Richardson, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reported+analgesic+and+anaesthetic+administration+to+rodents+undergoing+experimental+surgical+procedures.">Reported analgesic and anaesthetic administration to rodents undergoing experimental surgical procedures.</a> Lab Anim. 43, 149-154 (2009).</li><li>Flecknell, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laboratory Animal Anaesthesia - A Practical Introduction for Research Workers and Technicians. , (2009).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DIRECTIVE+2010/63/EU+OF+THE+EUROPEAN+PARLIAMENT+AND+OF+THE+COUNCIL+of+22+September+2010+on+the+protection+of+animals+used+for+scientific+purposes.">DIRECTIVE 2010/63/EU OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 22 September 2010 on the protection of animals used for scientific purposes.</a> Official Journal of the European Union. , (2010).</li><li>Geiger, B. M., Frank, L. E., Caldera-Siu, A. D., Pothos, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survivable+Stereotaxic+Surgery+in+Rodents.">Survivable Stereotaxic Surgery in Rodents.</a> J. Vis. Exp. (20), e880-e880 (2008).</li><li>Hellebrekers, L. J., Booij, L. H. D. J., Flecknell, P. A., Van Zutphen, L. F. M., Baumans, V., Beynen, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaesthesia,+analgesia+and+euthanasia.">Anaesthesia, analgesia and euthanasia.</a> Principles of Laboratory Animal Science. , 277-311 (2001).</li><li>Walantus, W., Elias, L., Kriegstein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+utero+intraventricular+injection+and+electroporation+of+E16+rat+embryos.">In utero intraventricular injection and electroporation of E16 rat embryos.</a> J. Vis. Exp. (6), e236-e236 (2007).</li><li>Paxinos, G., Watson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Rat Brain in Stereotaxic Coordinates. , (1986).</li><li>Paxinos, G., Watson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Rat Brain in Stereotaxic Coordinates. , (2007).</li><li>Swanson, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Brain Maps: Structure of the Rat Brain. , (1992).</li></ol>

作者什么都没有透露。

这个视频演示的主要目的是熟悉立体定向手术的基本原则的行为神经学家。研究人员已经进行立体定向手术，也可能从这个视频中受益，并考虑在自己的实验室中使用的一些程序上的修改。约1-3优化外科技术的不断增加知识，发展新的麻醉剂和止痛剂的使用在人用和兽用医药4,12，公众意识的动物福利问题，并严格的立法（例如，在2010年欧盟指令实验动物的使用 5）促使我...

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rodent stereotaxic surgery and animal welfare outcome improvements for behavioral neuroscience

立体定向手术植入到特定的大脑区域的套管已经几十年来，是一个非常成功的实验技术，以调查当地操纵的神经递质和信号转导通路中清醒，表现动物的影响。此外，立体植入电刺激电极和记录研究已有助于我们的神经可塑性和表现动物的大脑网络理解。外科技术在啮齿动物1-4，公众意识的动物福利问题，并严格的立法（例如，2010年欧盟指令对实验动物的使用 5）优化的知识不断增加，促使我们改进，特别是关于这些手术过程中，手术过程中实施新的程序，补充氧气和连续监测血氧和心率水平以及引入了手术后的护理的标准化协议。我们的观察表明，这些修改增加生存率和改善在手术后的动物的一般状况（如减肥和更积极的动物）。此视频演示将显示在此特别注意的若干修改的立体定向手术所涉及的一般程序。我们将说明这些大鼠的外科手术，但它也可以执行在老鼠或其他小实验动物，通过使用特殊的立体定位仪6适配器，这种类型的手术。

Watch this Scientific Journal Video about Rodent Stereotaxic Surgery and Animal Welfare Outcome Improvements for Behavioral Neuroscience at JoVE.com

Rodent Stereotaxic Surgery and Animal Welfare Outcome Improvements for Behavioral Neuroscience

对啮齿动物的立体定向手术允许的药物或电刺激和录音清醒，表现动物的有针对性的管理。在这个视频演示，我们将展示最新的程序上的修改这一长期的过程，成功地提高生存率和降低手术后的减肥。

啮齿动物的立体定向手术和动物福利成果改进行为神经科学

Stereotaxic surgery for the implantation of cannulae into specific brain regions has for many decades been a very successful experimental technique to ...

rodent-stereotaxic-surgery-animal-welfare-outcome-improvements-for

Research

JoVE Journal

Neuroscience

39.0K 次观看.  University of Groningen. 对啮齿动物的立体定向手术允许的药物或电刺激和录音清醒，表现动物的有针对性的管理。在这个视频演示，我们将展示最新的程序上的修改这一长期的过程，成功地提高生存率和降低手术后的减肥。

视频: Rodent Stereotaxic Surgery and Animal Welfare Outcome Improvements for Behavioral Neuroscience

Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution in assessing therapeutic efficacy. Between the two major classifications of administration, local vs. systemic, systemic delivery approaches are often preferred due to ease of administration. However, systemic delivery may result in suboptimal drug concentration being achieved in the CNS, and lead to erroneous conclusions regarding agent efficacy. Local drug delivery methods are more invasive, but may be necessary to achieve therapeutic CNS drug levels. Here, we demonstrate proper technique for three routes of systemic drug delivery: intravenous injection, intraperitoneal injection, and oral gavage. In addition, we show a method for local delivery to the brain: convection-enhanced delivery (CED). The use of fluorescently-labeled compounds is included for in vivo imaging and verification of proper drug administration. The methods are presented using murine models, but can easily be adapted for use in rats.

Thorough preclinical testing of drugs that act in the central nervous system often involves assessing and comparing drug biodistribution in association with specific routes of administration. Here, three commonly used methods of systemic delivery (intravenous, intraperitoneal, and oral) as well as a method for local delivery (convection-enhanced delivery) are demonstrated in mice.

全身和局部给药治疗的中枢神经系统疾病的啮齿动物模型

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution ...

科研

神经科学

Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in rodents have shown a direct link between testosterone and aggressive behavior1 and folk wisdom adapts these findings to humans, suggesting that testosterone induces antisocial, egoistic or even aggressive behavior2. However, many researchers doubt a direct testosterone-aggression link in humans, arguing instead that testosterone is primarily involved in status-related behavior3,4. As a high status can also be achieved by aggressive and antisocial means it can be difficult to distinguish between anti-social and status seeking behavior.
We therefore set up an experimental environment, in which status can only be achieved by prosocial means. In a double-blind and placebo-controlled experiment, we administered a single sublingual dose of 0.5 mg of testosterone (with a hydroxypropyl-&beta;-cyclodextrin carrier) to 121 women and investigated their social interaction behavior in an economic bargaining paradigm. Real monetary incentives are at stake in this paradigm; every player A receives a certain amount of money and has to make an offer to another player B on how to share the money. If B accepts, she gets what was offered and player A keeps the rest. If B refuses the offer, nobody gets anything. A status seeking player A is expected to avoid being rejected by behaving in a prosocial way, i.e. by making higher offers.
The results show that if expectations about the hormone are controlled for, testosterone administration leads to a significant increase in fair bargaining offers compared to placebo. The role of expectations is reflected in the fact that subjects who report that they believe to have received testosterone make lower offers than those who say they believe that they were treated with a placebo. These findings suggest that the experimental economics approach is sensitive for detecting neurobiological effects as subtle as those achieved by administration of hormones. Moreover, the findings point towards the importance of both psychosocial as well as neuroendocrine factors in determining the influence of testosterone on human social behavior.

The procedure described in this protocol shows that testosterone administration and folk beliefs about testosterone may be associated with directly opposed social behaviors.

睾酮和社会决策，结合行为内分泌和实验经济学:

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in ...

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

评估大鼠蔗糖渴求育成的一个通用方法

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Whole Animal Perfusion Fixation for Rodents

The goal of fixation is to rapidly and uniformly preserve tissue in a life-like state. While placing tissue directly in fixative works well for small pieces of tissue, larger specimens like the intact brain pose a problem for immersion fixation because the fixative does not reach all regions of the tissue at the same rate 5,7. Often, changes in response to hypoxia begin before the tissue can be preserved 12. The advantage of directly perfusing fixative through the circulatory system is that the chemical can quickly reach every corner of the organism using the natural vascular network. In order to utilize the circulatory system most effectively, care must be taken to match physiological pressures 3. It is important to note that physiological pressures are dependent on the species used. Techniques for perfusion fixation vary depending on the tissue to be fixed and how the tissue will be processed following fixation. In this video, we describe a low-cost, rapid, controlled and uniform fixation procedure using 4% paraformaldehyde perfused via the vascular system: through the heart of the rat to obtain the best possible preservation of the brain for immunohistochemistry. The main advantage of this technique (vs. gravity-fed systems) is that the circulatory system is utilized most effectively.

Here we describe a low-cost, rapid, controlled and uniform fixation procedure using 4% paraformaldehyde perfused via the vascular system: through the heart of the rat to obtain the best possible preservation of the brain.

整体动物灌注固定鼠害

The goal of fixation is to rapidly and uniformly preserve tissue in a life-like state. While placing tissue directly in fixative works well for small ...

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 ...

A Lightweight, Headphones-based System for Manipulating Auditory Feedback in Songbirds

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and sensorimotor plasticity1. Consistent sensory perturbations result in compensatory changes in motor output, reflecting changes in feedforward motor control that reduce the experienced feedback error. By quantifying how different sensory feedback errors affect human behavior, prior studies have explored how visual signals are used to recalibrate arm movements2,3 and auditory feedback is used to modify speech production4-7. The strength of this approach rests on the ability to mimic naturalistic errors in behavior, allowing the experimenter to observe how experienced errors in production are used to recalibrate motor output.
Songbirds provide an excellent animal model for investigating the neural basis of sensorimotor control and plasticity8,9. The songbird brain provides a well-defined circuit in which the areas necessary for song learning are spatially separated from those required for song production, and neural recording and lesion studies have made significant advances in understanding how different brain areas contribute to vocal behavior9-12. However, the lack of a naturalistic error-correction paradigm - in which a known acoustic parameter is perturbed by the experimenter and then corrected by the songbird - has made it difficult to understand the computations underlying vocal learning or how different elements of the neural circuit contribute to the correction of vocal errors13.
The technique described here gives the experimenter precise control over auditory feedback errors in singing birds, allowing the introduction of arbitrary sensory errors that can be used to drive vocal learning. Online sound-processing equipment is used to introduce a known perturbation to the acoustics of song, and a miniaturized headphones apparatus is used to replace a songbird's natural auditory feedback with the perturbed signal in real time. We have used this paradigm to perturb the fundamental frequency (pitch) of auditory feedback in adult songbirds, providing the first demonstration that adult birds maintain vocal performance using error correction14. The present protocol can be used to implement a wide range of sensory feedback perturbations (including but not limited to pitch shifts) to investigate the computational and neurophysiological basis of vocal learning.

We describe the design and assembly of miniaturized headphones suitable for replacing a songbird&#x2019;s natural auditory feedback with a manipulated acoustic signal. Online sound processing hardware is used to manipulate song output, introduce real-time errors in auditory feedback via the headphones, and drive vocal motor learning.

一个轻量级的，耳机为基础的系统操作鸣禽听觉反馈

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

在啮齿动物中枢神经系统的体内光遗传学刺激

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

新生小鼠脑巴细胞基因调控的立体定向手术

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

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.