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

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

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 environment^2,3, milk suckling^4-6, stro....

Protocol

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’s Institutional Animal Care Committee.

1. Nose Plug Construction

NOTE: This procedure was adapted and modified from Cummings et al. (1997)³⁵.

1. Aquire polyethylene-20 tubing and 3-0 silk suture thread.
2. Cut a small piece of polyethylene-20 tubing to approximately 0.8 mm.
3. Thread silk s....

Results

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 .......

Discussion

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 studies^33,34,36, 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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Polythylene 20 tubing	Intramedic	427406	Non-radiopaque, Non-toxic
3-0 Silk suture thread	Syneture	Sofsilk	Non-absorbent
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 brands should be okay too
Training box	Custom-made	N/A	Acrylic box (20 x 20 x 5 cm3), 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 (30 x 20 x 18 cm3), 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