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.

Sprague Dawley rat (Charles River) pups of both genders are used. Litters are culled to 12 on PD1 (birth being PD0). The dams are maintained on a 12 hr light/dark cycle with ad libitum access to food and water. Experimental procedures have been approved by Memorial University&#8217;s Institutional Animal Care Committee.
1. Nose Plug Construction
NOTE: This procedure was adapted and modified from Cummings et al. (1997)35.
<ol>
	<li>Aqui...

<ol>
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The authors have nothing to disclose.

The lateralized odor learning and memory model in rat pups within a critical time window was first established by Hall and colleagues. In a series of studies33,34,36, they showed that an odor preference memory could be lateralized by odor + milk pairings to one naris at PD 6 in rat pups. Preference memory was robust when the same naris was open during training and testing, but not observed when the occluded naris was unblocked and tested. However, at PD 12, when anterior commissural connections from the anteri...

Olfaction is the primary sensory modality in rodents, without which they would not be able to successfully navigate or survive in their environment. It is especially critical for neonatal pups, which can neither see nor hear during the first post-natal week, to use olfaction in order to locate their mother to feed1. As a result, neonatal rat pups can be conditioned to prefer odors with simple experimental manipulations. A variety of stimuli have been used as the unconditioned stimulus (UCS) to induce conditioned responses to novel odors (conditioned stimulus, CS) in neonates, including the nesting environment2,3, milk suckling4-6, stroking or tactile stimulation7-12, tail pinch13, maternal saliva13, mild foot shock14-18, and intracranial brain stimulation19. The present study employs a well-established early odor preference paradigm wherein an odor, in this case peppermint, is combined with tactile stimulation in order to produce a preference for peppermint 24 hr&#160;later10,11,20. These odors memories are dependent on intact olfactory circuitry, primarily including the olfactory bulbs (OB)21-23 and the anterior piriform cortex (aPC)24,25.
Experimental investigations of the early odor preference learning have deepened and broadened our understanding of the molecular and physiological underpinnings of a mammalian memory. This mammalian model has several advantages in studying memory mechanisms. First, the neural sources of the UCS signal have been identified. Various stimuli as mentioned above stimulate locus coeruleus norepinephrine release26, which in turn activates multiple adrenoceptors in the OB and aPC, causing cellular and physiological effects that support learning22,27,28. Second, memory-supporting mechanisms take place in well-defined laminar neural structures. The simplicity of the olfactory circuitry in neonatal rats provides researchers with the ideal framework with which to uncover the intricate processes related to synaptic plasticity. Olfactory sensory neurons (OSN) in the olfactory epithelium project onto mitral/tufted cells in the OB and these mitral/tufted cells in turn project ipsilaterally to piriform cortex (PC) via the lateral olfactory tract (LOT), among other structures29. Both the OSN synapses in the OB30,31 and the LOT synapses24,25 in aPC have been identified as critical loci for synaptic changes that support learning and memory. Third, in an early age in rats, olfactory inputs can readily be lateralized. Each aPC has access to bilateral odor information via the anterior commissure once this white matter is fully formed at post-natal day 12 (PD12)32. Before PD 12, odor input can be isolated to ipisilateral OB and aPC through single naris occlusion24,25,31,33,34. Single naris occlusion permits the odor memory formation from the open naris, and prevents the same memory from the occluded naris prior to PD 1233. Odor memory is isolated to the ipsilateral hemisphere including both OB and aPC. Therefore, each rat pup can be its own control for learning and underpinning physiology.
In the present study, the lateralized early odor preference learning protocol is introduced. This method serves as a powerful tool for studying neural mechanisms underpinning odor learning by providing an intra-animal control24,25,31 , thereby reducing both the number of animals required and the general variation. Naris occlusion is reversible in that the grease or nose plug can be applied and removed with minimal stress or damage to the animal. Here, first, detailed procedures of early odor preference training and testing are described, with a focus on the lateralized protocol using single naris occlusion with a nose plug. Then results are presented to demonstrate the effectiveness of single naris occlusion in isolating odor input and producing lateralized odor memory. Finally, the potentials of using this lateralized learning model to study physiological changes in the olfactory system that both generate learning and support memory expression are discussed.

<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.

Here we review some of the previously established results24 to demonstrate the effectiveness of the naris occlusion in isolating odor input and learning to one hemisphere, and the reversibility of this method.
Single naris occlusion during early odor preference training leads to a lateralized odor memory24. The memory is confined to the spared naris (Figure 3). When pups are tested for odor preference with the same naris occluded as during training, they ...

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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.

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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JoVE Journal

Neuroscience

8.8K ビュー.  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.