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>Aquire polyethylene-20 tubing and 3-0 silk suture thread.</li>
	<li>Cut a small piece of polyethylene-20 tubing to approximately 0.8 mm.</li>
	<li>Thread silk suture through the prepared tubing such that there is thread on either side of the section of tubing.</li>
	<li>On one end of the thread outside of the plug, tie a knot in the thread.</li>
	<li>Pull the section of tubing down over the knot in the thread. The knot should lodge inside the tubing.</li>
	<li>Trim both ends of the thread such that ~2 mm of thread is protruding from one end of the tubing (see Figure 1A).</li>
</ol>
2. Naris Occlusion before Training
<ol>
	<li>Remove pup from dam and place in a secure dish covered with regular bedding.</li>
	<li>Use a cotton tip application to dab a local anesthetic jelly, 2% Xylocaine, on the naris to be occluded.</li>
	<li>Allow the pup to rest in the dish for ~3 min.</li>
	<li>Hold the pup gently but securely in the non-dominant hand.</li>
	<li>Using the dominant hand, pick up a nose plug and dab the same local anesthetic jelly around the tip from which the thread is not protruding. This will act as both an anesthetic for any minor pains associated with plug insertion and will act as a lubricant inside the naris. NOTE: The effect of Xylocaine starts within a couple of minutes and lasts 20-30 min. Pups in general show good tolerance for plug insertion after xylocaine jelly application (minimum struggling and vocalization).</li>
	<li>Gently insert the nose plug by firmly holding the pup and slowly rotating the plug with very gentle pushes until the plug is fully inserted and only the 2 mm thread is protruding from the naris (see Figure 1B). There should be no bleeding from either naris during this process. Pups with bleeding during nose plug insertion are excluded and returned to dams</li>
	<li>Allow the animal to rest in this dish for 5 min&#160;in order to habituate to the plug.</li>
	<li>Remove the pup from the habituation dish and begin the conditioning paradigm24.</li>
</ol>
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51808/51808fig1highres.jpg" width="500" /> 
Figure 1.&#160;Construction of a nose plug. A)&#160;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)&#160;Front and lateral view of a rat with a nose plug in one naris.
3. Scented Bedding Preparation
<ol>
	<li>Wearing new gloves and in a fume hood to prevent odor contamination, place 500 ml of woodchip bedding into a plastic bag.</li>
	<li>Use a syringe to draw up 0.3 ml peppermint extract, and spray this over the bedding in the plastic bag.</li>
	<li>Tie the bag shut, shake the bag vigorously, and allow the bedding to rest in the bag for 5 min.</li>
	<li>Place the scented bedding in a clear, shallow, acrylic training box (20 x 20 x 5 cm3, Figure 2A) uncovered in a fume hood for 5 min&#160;before use. Once the bedding is prepared, discard these gloves, and do not allow these gloves to come in contact with the animals.</li>
	<li>Place the unscented bedding in an identical clear plastic box, and ensure that it does not come into contact with the scented bedding or used gloves.</li>
</ol>
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).
<ol>
	<li>Place the habituated pup on scented bedding. For control odor only (O/S-) pups, leave these pups on the bedding for 10 min, then skip to step 4.5. For experimental odor + stroke (O/S+) pups, continue to the following steps in this section.</li>
	<li>Stroke the pup for 30 sec&#160;using a small paintbrush. Use rapid circular motions primarily around the hind region of the pup.</li>
	<li>Allow the pup to rest for 30 sec.</li>
	<li>Repeat steps 4.2 &#38; 4.3 for a total of 10 min&#160;(i.e. 10 pairings of stroking + odor).</li>
	<li>Remove pup from the conditioning box, remove the nose plug and return the pup to the dam.</li>
</ol>
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).
<ol>
	<li>Prepare one peppermint and one unscented bedding as per Section 3, and place each box under opposite sides of the testing chamber, 2 cm apart. Place the plastic mesh on the metal-grid floor of the testing chamber.</li>
	<li>Remove the pup from the dam and place a firm dab of odorless silicone grease on the naris that is occluded during training. Re-apply the grease throughout the first testing procedure as needed. NOTE: Random naris occlusion during training and testing may be considered to avoid bias.</li>
	<li>Place pup in the neutral zone of the testing chamber.</li>
	<li>Allow the pup to explore the chamber for 1 min, recording how long the pup spent over the two sides of the chamber (i.e. over peppermint or neutrally-scented bedding).</li>
	<li>Allow the pup to rest for 1 min&#160;in a covered plastic holding chamber.</li>
	<li>Repeat steps 5.2 &#38; 5.3 for a total of 10 min&#160;(i.e. 5 test trials separated by 5 rest trials) switching the initial orientation of the pup in the chamber in order to control for direction preferences.</li>
	<li>Immediately following testing, wipe away the grease from the naris.</li>
	<li>Insert a polyethylene noseplug into the opposite naris as per section 2 and allow the animal to rest for 10 min.</li>
	<li>Test the pup once again as in 5.3 -5.6, remove the plug, and return the pup to the dam. Remove and clean the plastic mesh of the testing chamber with 95% ethanol and allow the liquid to evaporate. Place the mesh back before testing the next pup. 
	NOTE: Applying silicone grease at the first naris occlusion during testing prevents the chance of bleeding and stress associated with nose plug insertion.</li>
</ol>
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51808/51808fig2highres.jpg" width="500" /> 
Figure 2. Early odor preference training and testing. A)&#160;Early odor preference training using odor + stroking paradigm. B)&#160;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.
<ol>
	<li>Perform unilateral naris occlusions on PD 6 or 7 pups as described in section 2.</li>
	<li>After ~5 min&#160;habituation, place the pup in a covered plastic container and expose it to 30 &#181;l pure peppermint oil soaked in a piece of tissue for 10 min.</li>
	<li>Immediately after the peppermint odor exposure, inject the pup intraperitoneally (i.p.) with chloral hydrate (400 mg/kg) as a general anesthetic,&#160;or pentobarbitol, (150 mg/kg).</li>
	<li>Once fully anesthetized (showing no response to tail or foot pinch), transcardially perfuse the pup with ice-cold solutions of saline (0.9%) for ~1 min, followed by paraformaldehyde (4%, dissolved in 0.1 M phosphate buffer solution, PBS).</li>
	<li>After 10 min&#160;of paraformaldehyde perfusion, collect the brain and place it in paraformaldehyde overnight at 4&#160;&#176;C, then transfer the brain to a sucrose solution (20% in PBS) for an additional 24 hr.</li>
	<li>Cut coronal brain slices at 30 &#956;m thickness with a cryostat. Collect OB and PC slices and mount onto gelatin-coated slides, followed by standard immunohistochemistry staining for pCREB antibody21,25,30.</li>
</ol>
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.
<ol>
	<li>Perform unilateral naris occlusions on PD 6 or 7 pups as described in section 2.</li>
	<li>After 15 min&#160;(equivalent to the duration of the naris occlusion during training &#8211; 5 min&#160;habituation + 10 min&#160;training), remove the nose plug, and return the pup to the dam.</li>
	<li>24 hr&#160;later, expose the pup to the peppermint odor in a covered plastic container for 10 min&#160;as described in 6.2.</li>
	<li>Follow same steps in sections 6.3-6.6.</li>
</ol>

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+neural+representation+of+odorants+after+olfactory+deprivation+in+the+adult+mouse+olfactory+bulb.">Changes in the neural representation of odorants after olfactory deprivation in the adult mouse olfactory bulb.</a> Chem Senses. 38, 77-89 (2013).</li><li>Kim, H. H., Puche, A. C., Margolis, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant+deprivation+reversibly+modulates+transsynaptic+changes+in+the+NR2B-mediated+CREB+pathway+in+mouse+piriform+cortex.">Odorant deprivation reversibly modulates transsynaptic changes in the NR2B-mediated CREB pathway in mouse piriform cortex.</a> J Neurosci. 26, 9548-9559 (2006).</li><li>Korol, D. L., Brunjes, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+changes+in+2-deoxyglucose+uptake+and+amino+acid+incorporation+following+unilateral+odor+deprivation:+a+laminar+analysis.">Rapid changes in 2-deoxyglucose uptake and amino acid incorporation following unilateral odor deprivation: a laminar analysis.</a> Brain Res Dev Brain Res. 52, 75-84 (1990).</li><li>Leung, C. H., Wilson, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trans-neuronal+regulation+of+cortical+apoptosis+in+the+adult+rat+olfactory+system.">Trans-neuronal regulation of cortical apoptosis in the adult rat olfactory system.</a> Brain Res. 984, 182-188 (2003).</li></ol>

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><tbody><tr><td>Polythylene 20 tubing</td><td>Intramedic</td><td>427406</td><td>Non-radiopaque, Non-toxic</td></tr><tr><td>3-0 Silk suture thread</td><td>Syneture</td><td>Sofsilk</td><td>Non-absorbent</td></tr><tr><td>Silicone grease</td><td>Warner Instrument</td><td>64-0378</td><td>Odorless</td></tr><tr><td>2% Xylocaine gel</td><td>AstraZeneca</td><td>Prod. No 061</td><td>Lidocaine hydrochloride jelly, purchased at local pharmacy</td></tr><tr><td>Paint brush</td><td>Dynasty</td><td>206R</td><td>Similar size/other brands work too</td></tr><tr><td>Peppermint extract</td><td>Sigma-Aldrich</td><td>W284807</td><td>Other brands should be okay too</td></tr><tr><td>Training box</td><td>Custom-made</td><td>N/A</td><td>Acrylic box (20 x 20 x 5 cm&lt;sup&gt;3&lt;/sup&gt;), see &lt;b&gt;Figure 2A&lt;/b&gt;. 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><td>Testing chamber</td><td>Custom-made</td><td>N/A</td><td>Stainless steel (30 x 20 x 18 cm&lt;sup&gt;3&lt;/sup&gt;), see &lt;b&gt;Figure 2B&lt;/b&gt;. 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><td>pCREB antibody</td><td>Cell Signaling</td><td>9198</td><td>Ser 133 (87G3) Rabbit mAb</td></tr><tr><td>Chloral hydrate</td><td>Sigma-Aldrich</td><td>C8383</td><td>N/A</td></tr><tr><td>Paraformaldehype</td><td>Sigma-Aldrich</td><td>P6148</td><td>N/A</td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S9378</td><td>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 ...

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.

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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8.9K Views.  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.

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

The Double-H Maze: A Robust Behavioral Test for Learning and Memory in Rodents

Spatial cognition research in rodents typically employs the use of maze tasks, whose attributes vary from one maze to the next. These tasks vary by their behavioral flexibility and required memory duration, the number of goals and pathways, and also the overall task complexity. A confounding feature in many of these tasks is the lack of control over the strategy employed by the rodents to reach the goal, e.g., allocentric (declarative-like) or egocentric (procedural) based strategies. The double-H maze is a novel water-escape memory task that addresses this issue, by allowing the experimenter to direct the type of strategy learned during the training period. The double-H maze is a transparent device, which consists of a central alleyway with three arms protruding on both sides, along with an escape platform submerged at the extremity of one of these arms.
Rats can be trained using an allocentric strategy by alternating the start position in the maze in an unpredictable manner (see protocol 1; &#167;4.7), thus requiring them to learn the location of the platform based on the available allothetic cues. Alternatively, an egocentric learning strategy (protocol 2; &#167;4.8) can be employed by releasing the rats from the same position during each trial, until they learn the procedural pattern required to reach the goal. This task has been proven to allow for the formation of stable memory traces.
Memory can be probed following the training period in a misleading probe trial, in which the starting position for the rats alternates. Following an egocentric learning paradigm, rats typically resort to an allocentric-based strategy, but only when their initial view on the extra-maze cues differs markedly from their original position. This task is ideally suited to explore the effects of drugs/perturbations on allocentric/egocentric memory performance, as well as the interactions between these two memory systems.

The goal of this protocol is to investigate spatial cognition in rodents. The double-H water maze is a novel test, which is particularly useful to elucidate the different components of learning, consolidation and memory, as well as the interplay of memory systems.

Spatial cognition research in rodents typically employs the use of maze tasks, whose attributes vary from one maze to the next. These tasks vary by their ...

Behavior

A Method for Remotely Silencing Neural Activity in Rodents During Discrete Phases of Learning

This protocol describes how to temporarily and remotely silence neuronal activity in discrete brain regions while animals are engaged in learning and memory tasks. The approach combines pharmacogenetics (Designer-Receptors-Exclusively-Activated-by-Designer-Drugs) with a behavioral paradigm (sensory preconditioning) that is designed to distinguish between different forms of learning. Specifically, viral-mediated delivery is used to express a genetically modified inhibitory G-protein coupled receptor (the Designer Receptor) into a discrete brain region in the rodent. Three weeks later, when designer receptor expression levels are high, a pharmacological agent (the Designer Drug) is administered systemically 30 min prior to a specific behavioral session. The drug has affinity for the designer receptor and thus results in inhibition of neurons that express the designer receptor, but is otherwise biologically inert. The brain region remains silenced for 2-5 hr (depending on the dose and route of administration). Upon completion of the behavioral paradigm, brain tissue is assessed for correct placement and receptor expression. This approach is particularly useful for determining the contribution of individual brain regions to specific components of behavior and can be used across any number of behavioral paradigms.

This protocol describes how to temporarily and remotely silence neuronal activity in discrete brain regions while rats are engaged in learning and memory tasks. The approach combines pharmacogenetics (Designer-Receptors-Exclusively-Activated-by-Designer-Drugs) with a behavioral paradigm (sensory preconditioning) that is designed to distinguish between different components of learning.

This protocol describes how to temporarily and remotely silence neuronal activity in discrete brain regions while animals are engaged in learning and ...

Olfactory Context Dependent Memory: Direct Presentation of Odorants

Information is retrieved more effectively when the retrieval occurs in the same context as that in which the information was first encoded. This is termed context dependent memory (CDM). One category of cues that have been shown to effectively produce CDM effects are odors. However, it is unclear what the boundary conditions of these CDM effects are. In particular, given that olfaction has been called an implicit sense, it is possible that odors are only effective mnemonic cues when they are presented in the background. This assertion seems even more likely given that previous research has shown odors to be poor cues during paired associate memory tests, where odors are in the focus of attention as mnemonic cues for other information. In order to determine whether odors are only effective contextual mnemonic cues when presented outside the central focus of an observer, an olfactory CDM experiment was performed in which odorants were presented directly, rather than ambiently. Direct presentation was accomplished with the aid of an olfactometer. The olfactometer not only allows for direct presentation of odorants, but provides other methodological benefits, including the allowance of trial by trial manipulations of odorant presentations and, relatedly, time-specific releases of odorants. The presence of the same odor during both encoding and retrieval enhanced memory performance, regardless of whether the odor was presented ambiently or directly. This finding can serve as a basis for future olfactory CDM research which can utilize the benefits of direct presentation.

The use of an olfactometer for directly presenting odorants opens exciting opportunities for researchers of olfactory memory. The current paper discusses issues related to this methodology as related to a previously published experiment on olfactory context dependent memory.

Information is retrieved more effectively when the retrieval occurs in the same context as that in which the information was first encoded. This is termed ...

Appetitive Associative Olfactory Learning in Drosophila Larvae

In the following we describe the methodological details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with genetic interference, provides a handle to analyze the neuronal and molecular fundamentals of specifically associative learning in a simple larval brain.
Organisms can use past experience to adjust present behavior. Such acquisition of behavioral potential can be defined as learning, and the physical bases of these potentials as memory traces1-4. Neuroscientists try to understand how these processes are organized in terms of molecular and neuronal changes in the brain by using a variety of methods in model organisms ranging from insects to vertebrates5,6. For such endeavors it is helpful to use model systems that are simple and experimentally accessible. The Drosophila larva has turned out to satisfy these demands based on the availability of robust behavioral assays, the existence of a variety of transgenic techniques and the elementary organization of the nervous system comprising only about 10,000 neurons (albeit with some concessions: cognitive limitations, few behavioral options, and richness of experience questionable)7-10.
Drosophila larvae can form associations between odors and appetitive gustatory reinforcement like sugar11-14. In a standard assay, established in the lab of B. Gerber, animals receive a two-odor reciprocal training: A first group of larvae is exposed to an odor A together with a gustatory reinforcer (sugar reward) and is subsequently exposed to an odor B without reinforcement 9. Meanwhile a second group of larvae receives reciprocal training while experiencing odor A without reinforcement and subsequently being exposed to odor B with reinforcement (sugar reward). In the following both groups are tested for their preference between the two odors. Relatively higher preferences for the rewarded odor reflect associative learning - presented as a performance index (PI). The conclusion regarding the associative nature of the performance index is compelling, because apart from the contingency between odors and tastants, other parameters, such as odor and reward exposure, passage of time and handling do not differ between the two groups9.

Appetitive Associative Olfactory Learning in Drosophila Larvae

Drosophila larvae are able to associate odor stimuli with gustatory reward. Here we describe a simple behavioral paradigm that allows the analysis of appetitive associative olfactory learning.

In the following we describe the methodological  details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with ...

Drosophila Adult Olfactory Shock Learning

Drosophila have been used in classical conditioning experiments for over 40 years, thus greatly facilitating our understanding of memory, including the elucidation of the molecular mechanisms involved in cognitive diseases1-7. Learning and memory can be assayed in larvae to study the effect of neurodevelopmental genes8-10 and in flies to measure the contribution of adult plasticity genes1-7. Furthermore, the short lifespan of Drosophila facilitates the analysis of genes mediating age-related memory impairment5,11-13. The availability of many inducible promoters that subdivide the Drosophila nervous system makes it possible to determine when and where a gene of interest is required for normal memory as well as relay of different aspects of the reinforcement signal3,4,14,16.
Studying memory in adult Drosophila allows for a detailed analysis of the behavior and circuitry involved and a measurement of long-term memory15-17. The length of the adult stage accommodates longer-term genetic, behavioral, dietary and pharmacological manipulations of memory, in addition to determining the effect of aging and neurodegenerative disease on memory3-6,11-13,15-21.
Classical conditioning is induced by the simultaneous presentation of a neutral odor cue (conditioned stimulus, CS+) and a reinforcement stimulus, e.g., an electric shock or sucrose, (unconditioned stimulus, US), that become associated with one another by the animal1,16. A second conditioned stimulus (CS-) is subsequently presented without the US. During the testing phase, Drosophila are simultaneously presented with CS+ and CS- odors. After the Drosophila are provided time to choose between the odors, the distribution of the animals is recorded. This procedure allows associative aversive or appetitive conditioning to be reliably measured without a bias introduced by the innate preference for either of the conditioned stimuli. Various control experiments are also performed to test whether all genotypes respond normally to odor and reinforcement alone.

Drosophila Adult Olfactory Shock Learning

The method to measure adult Drosophila associative memory is described. The assay is based on the ability of the fly to associate an odor presented with a negative reinforcer (electric shock) and then recall this information at a later time, allowing memory to be measured.

Drosophila have been used in classical conditioning experiments for over 40 years, thus greatly facilitating our understanding of memory, including the ...

A Low-Cost, Odor-Reward Association Task for Tests of Learning and Memory

Robust and simple behavioral paradigms of appetitive, associative memory are crucial for researchers interested in cellular and molecular mechanisms of memory. In this paper, an effective and low-cost mouse behavioral protocol is described for examining the effects of physiological manipulation (such as the infusion of pharmacological agents) on the learning rate and duration of odor-reward memory. Representative results are provided from a study examining the differential role of tyrosine kinase receptor activity in short-term (STM) and long-term memory (LTM). Male mice were conditioned to associate a reward (sugar pellet) with one of the two odors, and their memory for the association was tested 2 or 48 h later. Immediately prior to the training, a tyrosine kinase (Trk) receptor inhibitor or vehicle infusions were delivered into the olfactory bulb (OB). Although there was no effect of the infusion on the learning rate, blockade of the Trk receptors in the OB selectively impaired LTM (48 h), and not short-term memory (STM; 2 h). The LTM impairment was attributed to the diminished odor selectivity as measured by the length of the digging time. The culmination of the results of this experiment showed that Trk receptor activation in the OB is the key in olfactory memory consolidation.

An odor-reward, associative learning task was used to investigate the differential effects of physiological manipulation on long-term and short-term memory.

Robust and simple behavioral paradigms of appetitive, associative memory are crucial for researchers interested in cellular and molecular mechanisms of ...

Designing a Flexible, Cost-Effective Olfactometer for Rodent Olfactory Experiments

Constructing an Olfactometer for Rodent Olfactory Behavior Studies

The use of olfactometers to study rodent behavior and brain activity during olfactory tasks is crucial for understanding brain circuits. These sophisticated devices allow researchers to precisely control and deliver odor stimuli, enabling the investigation of complex olfactory processes in rodents. Although commercially available olfactometers are convenient, they present challenges when technical issues arise, often requiring costly assistance and potentially disrupting research timelines. This article details the construction of a custom olfactometer specifically designed for mouse olfactory behavior experiments, providing a comprehensive list of parts and step-by-step instructions. The olfactometer is controlled through MATLAB, offering a user-friendly interface for researchers. Importantly, the open-source code allows users to modify and adapt the system, tailoring behavioral tasks to meet specific experimental needs. Building a customized olfactometer empowers users with the knowledge and capability to perform custom experimental design and troubleshooting independently, saving both time and resources. This approach not only enhances research flexibility but also fosters a deeper understanding of the equipment's functionality, ultimately leading to more robust and reliable olfactory studies in rodents.

Constructing an Olfactometer for Rodent Olfactory Behavior Studies Near-Infrared Spectroscopy Hyperscanning Study in Psychological Counseling

This protocol describes the construction of an olfactometer for go/no-go olfactory behavior experiments. Step-by-step instructions, along with images, are provided to ensure the successful construction of the olfactometer. Information for troubleshooting issues encountered during the process is also included.

The use of olfactometers to study rodent behavior and brain activity during olfactory tasks is crucial for understanding brain circuits. These ...

An Objective and Reproducible Test of Olfactory Learning and Discrimination in Mice

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and parenting. Importantly, mice can be trained to associate novel odors with specific behavioral responses to provide insight into olfactory circuit function. This protocol details the procedure for training mice on a Go/No-Go operant learning task. In this approach, mice are trained on hundreds of automated trials daily for 2&#8211;4 weeks and can then be tested on novel Go/No-Go odor pairs to assess olfactory discrimination, or be used for studies on how odor learning alters the structure or function of the olfactory circuit. Additionally, the mouse olfactory bulb (OB) features ongoing integration of adult-born neurons. Interestingly, olfactory learning increases both the survival and synaptic connections of these adult-born neurons. Therefore, this protocol can be combined with other biochemical, electrophysiological, and imaging techniques to study learning and activity-dependent factors that mediate neuronal survival and plasticity.

Here, we train mice on an associative learning task to test odor discrimination. This protocol also allows for studies on learning-induced structural changes in the brain.

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and ...

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously na&#239;ve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. ...

Aversive Associative Learning and Memory Formation by Pairing Two Chemicals in Caenorhabditis elegans

The nematode Caenorhabditis elegans is an attractive model organism to study learning and memory at molecular and cellular levels because of the simplicity of its nervous system, whose chemical and electrical wiring diagrams were completely reconstructed from serial electron micrographs of thin sections. Here, we describe detailed protocols for the conditioning of C. elegans by massed and spaced training for the formation of short-term memory (STM) and long-term memory (LTM), respectively. By pairing 1-propanol and hydrochloric acid as conditioned and unconditioned stimuli, respectively, C. elegans was successfully trained to form aversive associative STM and LTM. While na&#239;ve animals were attracted to 1-propanol, the trained animals were no longer or very weakly attracted to 1-propanol. Like in other organisms such as Aplysia and Drosophila, "learning and memory genes" play essential roles in memory formation. Particularly, NMDA-type glutamate receptors, expressed in only six pairs of interneurons in C. elegans, are required for the formation of both STM and LTM, possibly as a coincidence factor. Therefore, the memory trace may reside among the interneurons.

Aversive Associative Learning and Memory Formation by Pairing Two Chemicals in Caenorhabditis elegans

We have previously developed protocols for Caenorhabditis elegans to form short- and long-term associative memories by massed and spaced training, respectively. Here, detailed protocols are described for the conditioning of C. elegans by pairing 1-propanol and hydrochloric acid as conditioned and unconditioned stimuli, respectively, to form aversive associative memory.

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

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Chapters in this video

The Double-H Maze: A Robust Behavioral Test for Learning and Memory in Rodents

A Method for Remotely Silencing Neural Activity in Rodents During Discrete Phases of Learning

Olfactory Context Dependent Memory: Direct Presentation of Odorants

Appetitive Associative Olfactory Learning in Drosophila Larvae

Drosophila Adult Olfactory Shock Learning

A Low-Cost, Odor-Reward Association Task for Tests of Learning and Memory

Constructing an Olfactometer for Rodent Olfactory Behavior Studies

An Objective and Reproducible Test of Olfactory Learning and Discrimination in Mice

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Aversive Associative Learning and Memory Formation by Pairing Two Chemicals in Caenorhabditis elegans