This work was supported by a CIHR operating grant (MOP-102624) to Q. Y. We thank Dr Carolyn Harley for helpful discussions throughout the study, Dr. Qinlong Hou, Amin Shakhawat, and Andrea Darby-King for technical support.

无论男女，只SD大鼠（查尔斯河）的幼崽被使用。窝被扑杀，以12 PD1（出生是PD0）。水坝维持12小时的光/暗周期， 随意获取食物和水。实验步骤已通过纪念大学实验动物管理委员会。 1，鼻封堵施工注：此程序改编和卡明斯等人修改（1997）35。 <ol><li> AQUIRE聚乙烯20管和3-0丝线缝合线。 </li><li>剪一小块聚乙烯管材20到约0.8mm。 </li><li>螺纹丝缝线穿过准备管，使得有螺纹的管的部分的任一侧。 </li><li>在插头外螺纹的一端，打结的线。 </li><li>管拉下来了，在螺纹结的部分。结应提出管内。</li><li>修剪的螺纹，使得〜2毫米螺纹的从管的一端突出的两端（ 见图1A）。 </li></ol>训练前2娜丽丝闭塞<ol><li>在一个安全的菜铺上被褥定期删除大坝和地点的小狗。 </li><li>用棉签应用轻拍局部麻醉果冻，2％利多卡因，在鼻孔被遮挡。 </li><li>让小狗休息的菜〜3分钟。 </li><li>轻轻但稳固地握住小狗在非优势手。 </li><li>使用优势手，拿起一个鼻子插头并轻轻从该线程不突出的尖端周围的相同的局部麻醉果冻。这将作为这两种麻醉与插头插入相关的任何轻​​微疼痛，并会担任鼻孔内的润滑剂。注：利多卡因的效果开始不到两分钟，持续20-30分钟。幼仔一般表现出良好的耐受性的插头插入后利多卡因凝胶的应用（M挣扎的最小值和发声）。 </li><li>轻轻地紧紧抱着小狗，慢慢地旋转插头很温柔推，直到插头完全插入，只有2毫米螺纹从鼻孔插入伸出鼻子塞（ 见图1B）。应该有从任一鼻孔在此过程中没有出血。鼻子插头插入时有出血幼崽被排除在外，回到水坝</li><li>让动物休息的这盘5分钟才能到，观察者插头。 </li><li>取下习惯菜的小狗，并开始调节模式24。 </li></ol><img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51808/51808fig1highres.jpg" width="500" /> 图1。施工鼻子塞。 A）的示意图显示使鼻子塞的步骤。线程是通过聚乙烯管拉;一个结是由和PUL领进油管，以阻止它的中间;两端切有2mm残留在一端从管接头。 的B）前，用在一个鼻孔鼻插头大鼠的侧视图。 3，香味床上准备<ol><li>戴新手套，在通风橱，以防止异味污染，将500毫升木屑铺垫成一个塑料袋。 </li><li>用注射器吸取了0.3毫升薄荷提取物，并喷上这种在塑料袋中的床上用品。 </li><li>扎紧袋口封住，摇晃剧烈袋，并允许被褥休息收入囊中5分钟。 </li><li>将香味寝具清晰，浅，丙烯酸培训发现在通风橱中使用的前5分钟盒（20×20×5厘米3， 图2A）。一旦被褥准备，放弃这些手套，不要让这些手套来与动物接触。 </li><li>将无迹寝具相同的透明塑料盒，以及连接确保不接触到香味的被褥或使用的手套接触。 </li></ol> 4，气味调节范式（参见图图2A） 幼崽接受任何一个单一的空调会议上，PD 6或多个审判庭（每天一次会议，PD 3-6）。 <ol><li>广场上香气四溢的床上用品习惯于小狗。对于控制气味只有（O / S - ）的幼崽，离开被褥10分钟这些幼崽，然后跳到步骤4.5。对于实验异味+行程（O / S +）的幼崽，继续在本节下面的步骤。 </li><li>行程小狗30秒使用小画笔。使用快速打圈主要围绕着小狗的后腿区域。 </li><li>让小狗休息30秒。 </li><li>重复步骤4.2和4.3共10分钟（ 即 10配对行程+异味）。 </li><li>从调理盒中取出的小狗，取出鼻子塞和小狗回到大坝。 </li></ol> &lt;P舱=“jove_title”&gt; 5。单侧气味偏好测试（参见图图2B） 测试发生在不同时间点（ 例如 ，24或48小时）之后的最后的训练会话。试验是在一个不锈钢测试腔室（30×20×18 毫升 ），它被放置在两个训练拖曳（训练框在3.4中描述），以2厘米的中性区域分开顶部。一个培训箱含有薄荷香味的床上用品，而另一盒包含清洁，无味的床上用品。测试腔室的底板是金属网格，然后将其覆盖的塑料网状物的可移动片（ 图2B）。 <ol><li>制备1薄荷油和一种无味寝具按照第3章，并将其放置在测试室中，相距2厘米的相对侧上的每个方块。放在试验室的金属网格地板的塑料网。 </li><li>从大坝拆除的小狗和地点无味硅脂坚定民建联的鼻孔在训练中被遮挡。重新申请需要整个第一测试过程中润滑脂。注意：在训练和测试随机鼻孔闭塞，可以考虑，以避免偏差。 </li><li>放置小狗在测试室的中性区。 </li><li>让小狗探索室1分钟，记录多久的小狗花了室的两侧（ 即超过薄荷或中性香味的床上用品）。 </li><li>让小狗在一个带盖的塑料保持室休息1分钟。 </li><li>共10分钟，重复步骤5.2和5.3（ 即由5其余试验分离5试验试验）交换腔室中的幼犬的初始方向，以控制对方向的偏好。 </li><li>紧随其后的测试，擦去从鼻孔油脂。 </li><li>将聚乙烯noseplug到相反鼻孔按照第2节，让动物休息10分钟。 </li><li>再次测试的小狗在<html> 5.3 -5.6，拔下电源插头，并小狗回到大坝。除去和清洗试验室用95％乙醇的塑料网，并允许液体蒸发。测试下一小狗之前，将网格回来。 注意：在测试过程中的应用硅脂在第一鼻孔闭塞防止出血和鼻插头插入有关的应力的机会。 </li></ol><img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51808/51808fig2highres.jpg" width="500" /> 图2：早期气味偏好的培训和测试。一）早期气味偏好使用气味+抚摸着范式乙训练。）的一个侧面薄荷寝具两个选择气味偏好测试，控制无味的床上用品在另一侧。 2厘米中立区被放置在中间。 6，测试单娜丽丝闭塞的有效性NT“&gt;本实验的目的是确定单个鼻孔闭塞是否导致嗅觉系统的单侧激活。 <ol><li>在第2节中描述到PD 6或7的幼崽进行单侧鼻孔闭塞。 </li><li>后〜5分钟习惯，将幼鼠在有盖的塑料容器中，并暴露于30微升纯薄荷油浸泡在一块组织10分钟。 </li><li>紧接着的薄荷气味曝光，腹腔（IP）与水合氯醛（400毫克/千克）为全身麻醉，或戊巴比妥（150毫克/千克）注入小狗。 </li><li>一旦完全麻醉（示于尾部或脚捏无响应），transcardially灌注小狗用生理盐水（0.9％）为〜1分钟的冰冷溶液，随后的多聚甲醛（4％，溶于0.1M磷酸盐缓冲溶液，PBS ）。 </li><li>后的多聚甲醛灌注的10分钟，收集大脑，并放置在多聚甲醛中过夜，在4℃，然后反FER大脑到蔗糖溶液（在PBS中的20％），另外24小时。 </li><li>冠状切开大脑切片在30微米厚度与低温恒温器。收集转播和PC片和安装到明胶包被的载玻片，其次是标准的免疫组化染色区pCREB抗体21,25,30。 </li></ol> 7，测试单娜丽丝闭塞的可逆性该实验测试的阻挡效果是否是可逆的，在24小时之后除去鼻子塞。 <ol><li>在第2节中描述到PD 6或7的幼崽进行单侧鼻孔闭塞。 </li><li> 15分钟后（相当于鼻孔闭塞的持续时间期间训练 - 5分钟驯化+ 10分钟训练），除去鼻插头和小狗返回到坝。 </li><li> 24小时后，在6.2中描述揭露小狗的薄荷气味在有盖的塑料容器10分钟。 </li><li>按照教派相同的步骤离子6.3-6.6。 </li></ol>

<ol>
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W., Yuan, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+bulb+glomerular+NMDA+receptors+mediate+olfactory+nerve+potentiation+and+odor+preference+learning+in+the+neonate+rat.">Olfactory bulb glomerular NMDA receptors mediate olfactory nerve potentiation and odor preference learning in the neonate rat.</a> PLoS One. 7, e35024 (2012).</li><li>Yuan, Q., Harley, C. 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The authors have nothing to disclose.

首先由霍尔及其同事建立了幼鼠的单侧臭味学习和记忆模型中的一个关键的时间窗口。在一系列的研究中33,34,36，他们发现气味偏好的内存可以通过气味+豆浆配对的幼鼠进行单侧一个鼻孔在帕金森病6。偏好的记忆是强大的，当同样的鼻孔是在训练和测试开放的，但没有观察到，当闭塞鼻孔是畅通和测试。然而，在帕金森病12，当来自前嗅皮层前连合的连接成为功能32，未受过训练?...

嗅觉是初级感觉方式在啮齿类动物中，没有它们就不能成功地浏览或在其环境中生存。这是新生儿的幼崽，它可以既看不见也听不到在第一产后一周，用嗅觉，以找到他们的母亲喂养1尤其重要。因此，新生儿幼鼠能条件反射地偏爱气味与简单的实验操作。各种刺激已被用来作为非条件刺激（UCS）以诱导调节的反应，新颖的气味（条件刺激，CS）的新生儿，包括嵌套环境2,3，乳乳4-6，抚摸或触觉刺激7- 12，尾巴捏13，产妇的唾液13，轻度电击足底14-18，和颅内脑刺激19。本研究采用了一种行之有效的早期气味偏好的模式，其中的气味，在这种情况下薄荷，我š以产生偏爱薄荷24小时后10,11,20结合触觉刺激。这些气味的记忆是依赖于完整的嗅觉电路，主要包括嗅球（OB）21-23和前梨状皮质（APC）24,25。 早期的气味偏好的学习实验研究，加深和拓宽了我们的哺乳动物记忆的分子和生理基础的理解。这种哺乳动物模型具有学习记忆机制的几大优势。首先，UCS信号的神经来源已经确定。如上面提到的各种刺激刺激蓝斑的去甲肾上腺素释放26，从而激活多个肾上腺素能受体中的转播和APC，引起支持学习22,27,28细胞和生理效应。第二，存储器 - 支持机制发生在良好定义的层的神经结构。该新生大鼠嗅觉电路简洁为研究人员提供与发掘相关的突触可塑性的复杂过程的理想框架。嗅觉的感觉神经元（OSN）中的嗅觉上皮项目上二尖瓣/簇绒细胞中的转播和这些二尖瓣/簇绒细胞反过来项目同侧经由横向嗅束（LOT）梨状皮层（PC），其他结构29之间。无论是在OB 30,31的OSN突触和突触地段24,25参阅APC已被确定为关键位点为支持学习记忆的突触变化和。第三，在幼年大鼠，嗅觉输入可以很容易地单侧。每个APC有一次，通过这种白色物质完全形成，在产后第12天（PD12）32前联合访问两国的气味信息。帕金森病12日前，异味输入可隔离通过单鼻孔闭塞24,25,31,33,34 &lt;到ipisilateral转播和APC/ SUP&gt;。单鼻孔闭塞允许从开启鼻孔的气味记忆的形成，并且防止在同一存储器从现有的闭塞鼻孔到PD 12 33。气味存储器分离到同侧半球包括OB和APC。因此，每只大鼠的小狗可以自己控制学习和支撑生理学。 在本研究中，单侧早期气味偏好学习协议被引入。该方法可作为一种强大的工具用于研究通过提供一种帧内动物控制24,25,31，从而减少了所需的动物数量和一般的变异支撑气味学习神经机制。鼻孔闭塞是可逆的，所述润滑脂或鼻子塞可以应用和以最小的应力或者损坏的动物中移除。在这里，首先，早期气味偏好的培训和测试的详细过程进行了描述，重点是使用单鼻孔闭塞了号的单侧协议ê插头。然后结果被提出来证明在分离的气味输入并产生单侧气味存储器单鼻孔闭塞的有效性。最后，利用该单侧学习模型来研究生理变化的嗅觉系统，这两个生成学习和支撑记忆表达的潜力进行了讨论。

<table border="0" cellpadding="0" cellspacing="0" width="580">
	<tbody>
		<tr height="50">
			<td height="50" style="height:50px;width:149px;">Polythylene 20 tubing</td>
			<td style="width:123px;">Intramedic</td>
			<td style="width:103px;">427406</td>
			<td style="width:205px;">Non radiopaque, Non toxic</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:149px;">3-0 silk suture thread</td>
			<td style="width:123px;">Syneture</td>
			<td style="width:103px;">Sofsilk</td>
			<td style="width:205px;">Non absorbant&#160;</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:149px;">Silicone grease</td>
			<td style="width:123px;">Warner Instrument</td>
			<td style="width:103px;">64-0378</td>
			<td style="width:205px;">Odorless</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:149px;">2% xylocaine gel</td>
			<td style="width:123px;">AstraZeneca</td>
			<td style="width:103px;">Prod. No 061</td>
			<td style="width:205px;">Lidocaine hydrochloride jelly,&#160; purchased at local pharmacy</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:149px;">Paint brush</td>
			<td style="width:123px;">Dynasty</td>
			<td style="width:103px;">206R</td>
			<td style="width:205px;">Similar size/other brands work too</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:149px;">Peppermint extract</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:103px;">W284807</td>
			<td style="width:205px;">Other brand should be okay too</td>
		</tr>
		<tr height="171">
			<td height="171" style="height:171px;width:149px;">Training box</td>
			<td style="width:123px;">Custom-made</td>
			<td style="width:103px;">N/A</td>
			<td style="width:205px;">Acrylic box (20x20x5cm3), see Figure 2A. Parameters and material for the box are not critical and can be modified. Material used should be odorless and does not absorb odors</td>
		</tr>
		<tr height="182">
			<td height="182" style="height:182px;width:149px;">Testing chamber</td>
			<td style="width:123px;">Custom-made</td>
			<td style="width:103px;">N/A</td>
			<td style="width:205px;">Stainless steel (30x20x18cm3), see Figure 2B. Parameters and material for the chamber are not critical and can be modified. For example, an acrylic chamber instead of a stainless steel one can be used</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:149px;">pCREB antibody</td>
			<td style="width:123px;">Cell Signaling</td>
			<td style="width:103px;">9198</td>
			<td style="width:205px;">Ser 133 (87G3) Rabbit mAb</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:149px;">Chloral hydrate</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:103px;">C8383</td>
			<td style="width:205px;">N/A</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:149px;">Paraformaldehype</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:103px;">P6148</td>
			<td style="width:205px;">N/A</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:149px;">Sucrose</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:103px;">S9378</td>
			<td style="width:205px;">N/A</td>
		</tr>
	</tbody>
</table>

a lateralized odor learning model in neonatal rats for dissecting neural circuitry underpinning memory formation

Rat pups during a critical postnatal period (&#8804; 10 days) readily form a preference for an odor that is associated with stimuli mimicking maternal care. Such a preference memory can last from hours, to days, even life-long, depending on training parameters. Early odor preference learning provides us with a model in which the critical changes for a natural form of learning occur in the olfactory circuitry. An additional feature that makes it a powerful tool for the analysis of memory processes is that early odor preference learning can be lateralized via single naris occlusion within the critical period. This is due to the lack of mature anterior commissural connections of the olfactory hemispheres at this early age. This work outlines behavioral protocols for lateralized odor learning using nose plugs. Acute, reversible naris occlusion minimizes tissue and neuronal damages associated with long-term occlusion and more aggressive methods such as cauterization. The lateralized odor learning model permits within-animal comparison, therefore greatly reducing variance compared to between-animal designs. This method has been used successfully to probe the circuit changes in the olfactory system produced by training. Future directions include exploring molecular underpinnings of odor memory using this lateralized learning model; and correlating physiological change with memory strength and durations.

在这里，我们回顾一些先前建立的结果24，以显示在分离的气味输入和学 ​​习到一个半球的鼻孔闭塞的有效性，并且该方法的可逆性。 在早期的气味偏好训练单鼻孔闭塞导致单侧气味记忆24。该存储器被限制在幸免鼻孔（ 图3）。当幼崽进行了测试气味的喜好与同鼻孔闭塞的训练时，他们表现出偏爱空调异味（ 如薄荷）。当幼崽与封闭相...

Watch this Scientific Journal Video about A Lateralized Odor Learning Model in Neonatal Rats for Dissecting Neural Circuitry Underpinning Memory Formation at JoVE.com

A Lateralized Odor Learning Model in Neonatal Rats for Dissecting Neural Circuitry Underpinning Memory Formation

This protocol introduces lateralized early odor preference learning in rats using acute single naris occlusion. Lateralized learning permits the examination of behavioral outcomes and underpinning biological mechanisms within the same animals, reducing variance induced by between-animal designs. This protocol can be used to investigate molecular mechanisms underpinning early odor learning.

新生大鼠于A气味偏侧学习模型解剖神经回路托换记忆的形成

Rat pups during a critical postnatal period (&#8804; 10 days) readily form a preference for an odor that is associated with stimuli mimicking maternal ...

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Neuroscience

8.8K 次观看.  Faculty of Medicine, Memorial University. This protocol introduces lateralized early odor preference learning in rats using acute single naris occlusion. Lateralized learning permits the examination of behavioral outcomes and underpinning biological mechanisms within the same animals, reducing variance induced by between-animal designs. This protocol can be used to investigate molecular mechanisms underpinning early odor learning.