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
Summary
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.
Abstract
Rat pups during a critical postnatal period (≤ 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.
Introduction
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 <到ipisilateral转播和APC/ SUP>。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。
Protocol
Representative Results
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Polythylene 20 tubing | Intramedic | 427406 | Non radiopaque, Non toxic |
| 3-0 silk suture thread | Syneture | Sofsilk | Non absorbant |
| Silicone grease | Warner Instrument | 64-0378 | Odorless |
| 2% xylocaine gel | AstraZeneca | Prod. No 061 | Lidocaine hydrochloride jelly, purchased at local pharmacy |
| Paint brush | Dynasty | 206R | Similar size/other brands work too |
| Peppermint extract | Sigma-Aldrich | W284807 | Other brand should be okay too |
| Training box | Custom-made | N/A | 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 |
| Testing chamber | Custom-made | N/A | 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 |
| pCREB antibody | Cell Signaling | 9198 | Ser 133 (87G3) Rabbit mAb |
| Chloral hydrate | Sigma-Aldrich | C8383 | N/A |
| Paraformaldehype | Sigma-Aldrich | P6148 | N/A |
| Sucrose | Sigma-Aldrich | S9378 | N/A |
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