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 (≤ 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.
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 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.
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.
2. Naris Occlusion before Training
Figure 1. Construction of a nose plug. A) Schematics showing the steps of making a nose plug. A thread is pulled through polyethylene tubing; a knot is made and pulled into the middle of the tubing to block it; two ends are cut with a 2 mm residue in one end out of the tubing. B) Front and lateral view of a rat with a nose plug in one naris.
3. Scented Bedding Preparation
4. Odor Conditioning Paradigm (See Picture in Figure 2A)
Pups undergo either a single conditioning session, on PD 6, or multiple trial sessions (one session per day, PD 3-6).
5. Lateralized Odor Preference Testing (See Picture in Figure 2B)
Testing occurs at various time points (e.g., 24 or 48 hr) following the final training session. Testing is carried out in a stainless steel testing chamber (30 x 20 x 18 cm3), which is placed on top of two training boxes (training box is described in 3.4), separated by a 2 cm neutral zone. One training box contains peppermint-scented bedding while the other box contains clean, unscented bedding. The floor of the testing chamber is a metal grid, which is then covered by a removable sheet of plastic mesh (Figure 2B).
Figure 2. Early odor preference training and testing. A) Early odor preference training using odor + stroking paradigm. B) Two choice odor preference testing with peppermint bedding on one side, control unscented bedding on the opposite side. A 2 cm neutral zone is placed in between.
6. Testing the Effectiveness of Single Naris Occlusion
This experiment is performed to determine whether single naris occlusion leads to lateralized activation of the olfactory system.
7. Testing the Reversibility of Single Naris Occlusion
This experiment tests whether the blocking effect is reversible at 24 hr following the removal of the nose plug.
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 show preference for the conditioned odor (e.g., peppermint). When pups are tested with the opposite naris occluded, they show no preference for the conditioned odor. Together, these results suggest that an odor preference memory is only formed and expressed through the spared naris that underwent the odor + stroking associative conditioning.
Figure 3. Single naris occlusion induces lateralized odor learning. The behavioral protocol is shown in the upper panel. Odor + stroking (O/S+) or odor only (O/S–) animals with single naris occluded during training, underwent odor preference testing first with the same naris occluded then with the opposite naris occluded. The lower panel shows the percentage of time spent over peppermint-scented bedding among different groups in a two choice odor test. *p < 0.05. Error bars, mean ± SEM. Reproduced from Fontaine et al. J. Neuroscience (2013) with permission.
The lateralized odor training results in lateralized activation of the olfactory system during odor exposure (Figure 4)24. Single naris occlusion prevents activation of the OB and the PC of the ipsilateral hemisphere during odor exposure. This is demonstrated by monitoring CREB phosphorylation in the OB, and the PC. As shown in Figure 4, using immunohistochemistry, phosphorylated CREB (pCREB) is significantly less in the occluded hemisphere following peppermint odor exposure, compared to the contralateral spared hemisphere. Nissl staining (Figure 4A) demonstrates comparable cell bodies in the mitral cell layer of the OB, and in the pyramidal cell layer of the PC of both hemispheres. However, pCREB is significantly less in both cell layers in the hemisphere ipsilateral to the occluded naris (Figure 4B).
Figure 4. Single naris occlusion results in lateralized activation of the olfactory system during odor exposure. A) Nissl staining of the olfactory bulb (OB) and anterior piriform cortex (aPC). B) PCREB expressions in the occluded and spared hemispheres following peppermint exposure in a single naris occluded pup. Arrows indicate mitral cell layer in the OB and pyramidal cell layer in the aPC. Scale bars, 500 μm. Reproduced from Fontaine et al. J. Neuroscience (2013) with permission.
The effect of a single trial (15-20 min) naris occlusion is transient and reversible, and does not result in visible longer-term neuronal damage that could lead to altered odor perception and reduced neuronal activation to odors during testing24. As indexed by pCREB staining in the OB and the PC (Figure 5), pCREB expressions in mitral cells of the OB, and the pyramidal cells in the PC to odor exposure are comparable between the occluded and spared hemispheres, 24 hr following the removal of the nose plug – the same time point that odor preference testing is carried out following early odor preference training.
Figure 5. Assessment of neuronal reactivity following reversible naris occlusion. PCREB staining of the OB and the aPC, 24 hr following the removal of a nose plug in one pup. Arrows indicate mitral cell layer in the OB and pyramidal cell layer in the aPC. MCL, mitral cell layer. PCL, pyramidal cell layer. Scale bars, 500 µm for lower magnification and 100 μm for higher magnification.
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 anterior olfactory cortices become functional32, the untrained naris alone could support the expression of the odor preference acquired at PD 6. Lesion of the anterior commissure restored the lateralized memory so it was no longer accessible from the untrained naris33. This lateralized learning model has been successfully replicated with an odor + stroking paradigm24,25,31. Odor + stroking training with single naris occlusion on PD 6 leads to a lateralized memory on PD 731. Repeated single naris occlusions on PD 3-6 leads to a longer lateralized memory that lasts at least 48 hr24.
The lateralized odor learning protocol results in “split brain” in individual animals. This model has great advantages for studying behavior and the underpinning bio-physiological changes. For instance, comparing behavioral outputs using a within-subject model sufficiently reduces variations in between-animal designs. Pups at this early age vary considerably in their activity and responsiveness. Intra-animal control removes the inherent individual variability in performance and responsiveness as well as intrinsic differences in biology from the assessment of molecular and physiological changes. Additionally, this powerful infant model of lateralized learning lends us an opportunity to relate individual memory performance to individual physiology, and to assess the underpinnings of memories of different durations24,25,31. Using this lateralized learning model in combination with ex vivo experiments comparing the physiology of the two hemispheres within the same animals, It has been recently demonstrated that early odor learning induces synaptic plasticity such as increased AMPA receptor responses at synapses in both the OB31, and the aPC24,25. Enhanced synaptic transmission following early odor learning translates into an enhanced output in olfactory network representations24.
Future studies should explore the molecular underpinnings of odor memory using this lateralized learning model. This includes correlational studies looking at proteins and genes activated following learning, and causal studies looking at the effects of gain-of-function and loss-of-function of proteins and genes of particular interest. Another exciting and important possibility is to be able to relate physiological and molecular changes to the strength of behavioral memory. For each pup, it is possible to first derive a preference memory measure for the open and occluded nares. Subsequent ex vivo experiments on the corresponding trained and untrained cortices would provide correlative physiological changes. It is possible, however, that odor re-exposure during preference testing itself will alter synaptic strength, or that other brain regions contribute significantly to memory expression. In our present studies, physiology and behavior are carried out on separate cohorts. This removes concerns about behavioral testing influencing the very parameters of interest.
One caveat using nose plug is potential neural tissue damage that is associated with plug insertion and removal. For this reason, care should been taken in insertion of the nose plug and pups with bleeding during insertion should be excluded, to avoid any potential longer-term effects associated with bleeding such as inflammation. Prolonged blockage (hours, days to months) of naris or permanent ablation of olfactory epithelium leads to long-term olfactory deprivation, neural damage, and reduced neuronal activities in the OB and PC37-41, even though some of these effects due to prolonged naris occlusion are fully reversible39,40. Tissue integrity following acute naris occlusion (~15 min) has been validated by immunohistochemistry staining of pCREB, an activity-dependent neuronal marker, which has been shown to be reduced in the PC of young adult mice following 5-day naris occlusion39. pCREB levels in the ipsilateral OB and PC of the occluded naris were significantly less during peppermint odor exposure, confirming the successful lateralization of olfactory throughput in rat pups during naris occlusion. However, 24 hr following the removal of the nose plug, at the time of odor preference testing, the pCREB levels are comparable in both hemispheres, suggesting the effect of acute occlusion is fully reversible24. Therefore, the lack of preference to peppermint tested with the previously occluded naris is due to lack of memory, but not due to an alteration or lack of odor perception associated with tissue damage during testing. Additionally, electrophysiological recordings of control O/S– animals (with one naris occluded during odor exposure without stroking) showed no differences in the fEPSPs or number of activated pyramidal cells seen with calcium imaging – also confirming there is no functional change in the piriform cortex due to these short-term reversible naris occlusions24,25.
The authors have nothing to disclose.
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.
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 |