The authors have no acknowledgements.

All data collection in the original experiment15 was performed in accordance with the ethical guidelines of the American Psychological Association as well as the World Medical Association&#8217;s Declaration of Helsinki. The experiment was preapproved by the local Ethik-Kommission (equivalent to an Institutional Review Board). All subjects were made aware that their participation was voluntary and could at any time and without fear of penalty be terminated. All subjects signed informed consent.
1. Participant Recruitment and Pre-study Instructions
<ol>
	<li>Recruitment:
	<ol>
		<li>Recruit participants who are native speakers (to ensure a comparable understanding of the instructions and comparable processing of target information) and who have normal or corrected to normal vision. Also, either a) exclude subjects who profess below average olfactory abilities or are smokers, or b) collect information about smoking habits and olfactory ability to use in later analyses.</li>
		<li>Furthermore, given the frequency of sex differences found in olfactory studies18, either a) recruit equal numbers of males and females, to allow for comparisons, b) perform analyses separately for each sex to ensure no differences, or c) focus on a single sex. Finally, given that olfactory performance is age dependent19 either a) recruit all subjects from a single cohort, or b) specifically include age as a variable of interest. 
		Note: The final sample used in the current study included 60 subjects (56 females, Mage = 22.05 years, SD = 4.86), all of whom were students at the University of Hildesheim and who participated in exchange for partial course credit. Further information concerning the sample can be found in our previous article15.</li>
	</ol>
	</li>
	<li>Provide subjects with specific pre-study instructions. 1) Subjects must not smoke for the 24 h prior to the beginning of the experiment, 2) Subjects must not eat or brush teeth for at least one hour prior to the beginning of the experiment.</li>
	<li>When subjects arrive in the laboratory ensure that they have followed the pre-study instructions. This is best accomplished by having the experiment leader specifically ask the subjects whether they have complied with the instructions.</li>
	<li>If they have complied, present subjects with information about the study and any ethical considerations. Have subjects sign informed consent.</li>
	<li>Situate subjects in the experimental room. 
	NOTE: In the current experiment subjects were first placed in a cabin in which there was a monitor rested upon a table.
	<ol>
		<li>Seat subjects in a comfortable chair which they can adjust as needed. Have subjects place their chins in a chin rest (in which the tubes for presenting the odors are located). Adjust the height of the chin rest to make the seating comfortable for the subject.</li>
		<li>Alternatively, use another method of presentation, including placing the tubes directly in the nostrils of the subjects or connecting the tube-ends to a mask, which ensures that the odorous material remains concentrated around the nose.</li>
	</ol>
	</li>
</ol>
2. Experimental Design
NOTE: In the experiment outlined here (which corresponds to Experiment 1 from the original article15) the purpose was to determine whether the presence of an odor context during encoding and retrieval enhanced memory over a condition in which no extra odor context was present during both phases5. This is a variety of a CDM effect called context enhanced memory. The original experiment included more independent variables (odor valence and word valence) that are irrelevant for the current report. For details on the original experiment and specific information concerning the verbal material used, see the Supplementary Material from the original article15. The experiment is broken down into three different phases (See Table 1 for an overview).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Pre-study</td>
			<td>Encoding phase</td>
			<td>Retention interval</td>
			<td>Retrieval phase</td>
		</tr>
		<tr>
			<td>1. Ensure pre-study instructions were follwed</td>
			<td>1. Present words one at a time</td>
			<td>1. 5 minute break in which subjects color in mandalas</td>
			<td>1. Present words one at a time</td>
		</tr>
		<tr>
			<td>2. Have subjects sign informed-concsent</td>
			<td>2. Subjects complete abstract/concrete categorization task</td>
			<td></td>
			<td>2. Subjects complete old/new recognition test</td>
		</tr>
		<tr>
			<td>3. Situate subjects in experimental room</td>
			<td>3. Present odorants on each trial</td>
			<td></td>
			<td>3. Present odorants on each trial</td>
		</tr>
		<tr>
			<td>4. Perform practice inhalation trials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Overview of the experimental procedure.
<ol>
	<li>For the first, encoding, phase have subjects encode the target material (words) in the presence of the contextual material (odors).
	<ol>
		<li>Present subjects with words (one at a time) on the screen. Subjects must categorize the words as abstract or concrete. Learning is incidental, as subjects are not informed of the later memory test.</li>
		<li>Have subjects categorize 90 words in total.</li>
		<li>Have each trial follow the same pattern.
		<ol>
			<li>Begin each trial with the presentation of a puff of air. The puff of air either contains odorous material or not, depending on the group.</li>
			<li>Couple the puff of air the presentation of a cross on the computer screen and have the puff of air and the cross remain for 2,000 ms.</li>
			<li>Replace the cross is replaced with a target word, and have it remain for 2,000 ms. Order word presentation randomly for each subject.</li>
			<li>Upon offset of the word, have subjects categorize the word as abstract or concrete by button press. The response triggers the immediate onset of the next trial. Present the same odorant on all 90 trials during the encoding phase.</li>
		</ol>
		</li>
		<li>Odor presentation.
		<ol>
			<li>Utilize an olfactometer to achieve direct presentation of odorants. Olfactometers come in several different varieties. Purchase or build an olfactometer that will best meet the aims of the researchers25.</li>
			<li>Ensure that the olfactometer design is appropriate to the research question.</li>
			<li>Cut the tubes to the appropriate length and ensure that they are held in place and tightly connected. 
			NOTE: The length of the tubes influences the delay between signal onset (i.e., signal from program to release an odorant) and actual arrival of odorous material to the nostrils. Researchers should be aware of the delays between signals and actual presentation times, which will vary between individual olfactometers. This information can usually be found in information provided by the retailer or in instructions for building new olfactometers.</li>
			<li>Be aware of potential issues involved with odorant presentation.
			<ol>
				<li>Ensure that the inter-stimulus interval (ISI; or the length of time between odorant presentations) is appropriate for the research question. There is no standardized or suggested ISI appropriate for all odor experiments. Conduct pilot tests to determine which ISI&#8217;s are appropriate for the experiment in question.</li>
				<li>Control for tactile stimulation. Present the tubes inside a metal bar that supports the chin rest. Have the tubes end approximately 2 cm below the nostrils. Cover the top of the metal bar with a filter (e.g., tissue). Replace the filter for each subject.</li>
				<li>Control for auditory stimulation. Have subjects perform the experiment inside a sound-reducing cabin with headphones on. Store the olfactometer itself in a sound-reducing container unit stored outside of the experimental cabin.</li>
			</ol>
			</li>
		</ol>
		</li>
		<li>For presentation control, use a standard experimental control software program (see Table of Materials) to control odorant presentation from the olfactometer.</li>
		<li>Breathing instructions. 
		NOTE: When using an olfactometer, it is potentially important that subjects sniff (i.e., sample the olfactory material) at the appropriate moment. This is particularly important in experiments where the odorant changes between trials.
		<ol>
			<li>To ensure that subjects sample the odorant at the appropriate time, present a visual cue (fixation cross) simultaneously with the onset of the puff of air. Furthermore, ensure that the puff of air is long enough to allow the subjects to register the visual cue and initiate inhalation (2,000 ms). It should be noted here that the inhalation instruction might act as a second task, which could potentially interact with the task(s) of interest.</li>
			<li>Alternatively, use one of several other methods for ensuring subjects sample the odorants at the appropriate time:
			<ol>
				<li>Present subjects with a countdown, so they can prepare to inhale when the countdown is completed</li>
				<li>Present subjects with an initial cue alerting them to exhale followed by a second cue alerting them to inhale</li>
				<li>If the tubes are presented directly in the nostrils, and a discrete presentation olfactometer is used, instruct subjects to inhale anytime they feel tactile stimulation inside the nostrils.</li>
			</ol>
			</li>
			<li>Regardless of which method is used, have subjects complete inhalation practice trials before beginning with the actual experiment.</li>
			<li>For practice trials, instruct subjects to inhale whenever they see the appearance of the fixation cross. Present a fixation cross and an air puff for 2,000 ms on each trial. Have subjects complete 10 practice trials.</li>
		</ol>
		</li>
		<li>Timing.
		<ol>
			<li>Present the odorous material two seconds prior to the onset of the visually presented word. This is done to ensure that the olfactory context is present during visual perception.</li>
			<li>Other research questions may call for simultaneous perception. Olfactory perception takes longer than visual and auditory perception from the time of stimulus onset until perception20, at least as measured by response times on detection tasks21. Therefore, depending on the manipulation, researchers must ensure that they choose appropriate inter-stimulus intervals (ISIs). Note that this will also depend on the specifics of the olfactometer used (see 4.1.)</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>For the second phase, use a retention interval in which subjects complete a task that is unrelated to the actual memory task.
	<ol>
		<li>Have subjects leave the testing cabin and move to a different area, away from odorous material. This is an important step to ensure that the context is reinstated during the retrieval phase.</li>
		<li>Air out the testing cabin during the retention interval with ventilators or fans.</li>
		<li>Have subjects color in mandalas for 5 minutes. 
		NOTE: OCDM experiments have frequently, though not always14, utilized long retention intervals to avoid issues of adaptation and habituation. One potential benefit of direct presentation of discrete air puffs is that it may be more resistant to habituation than ambient presentation.</li>
		<li>Alternative distractor tasks can be implemented in the retention interval to accomplish different goals.</li>
	</ol>
	</li>
	<li>For the third, retrieval phase, have subjects remember and retrieve the target information.
	<ol>
		<li>Have subjects return to the experimental cabin. Once again, seat subjects with their chins in the chin rest.</li>
		<li>Have subjects complete an old/new recognition test with a total of 180 trials (90 old/90 new).</li>
		<li>Have each trial follow the same pattern.
		<ol>
			<li>Begin each trial with the presentation of a puff of air. The puff of air either contains odorous material or not, depending on the group. As this is a context enhancement experiment, the odorant presented is always the same as that presented in the encoding phase.</li>
			<li>Couple the puff of air with the presentation of a cross on the computer screen; have both the puff of air and the cross remain for 2,000 ms.</li>
			<li>Replace the cross with a word and have the subject respond (by button press) as to whether the word is old or new. The word remains until the subject responds. The order of word presentation is determined randomly for each subject.</li>
			<li>Have the subject&#8217;s response trigger the immediate onset of the next trial. There are no time limits during the retrieval phase.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Stimulus Selection
<ol>
	<li>Target stimuli
	<ol>
		<li>Select target stimuli that best fit the exact research question or the type of memory test that the experimenter is planning. For the most basic design, choose stimuli that come from a non-olfactory modality (e.g., words). For more information about the target stimuli used in the experiment reported here see the original article15.</li>
	</ol>
	</li>
	<li>Contextual stimuli
	<ol>
		<li>Select odorants that best fit the exact research question. Important attributes to take into account include 1) valence, 2) arousal, 3) intensity, 4) familiarity, and 5) identifiability.</li>
		<li>There are several methods that can be used when choosing the appropriate odorants.
		<ol>
			<li>In one method, choose the odorants based on a pilot study. In this case, have external subjects (those not participating in the main experiment) rate a variety of potential odorants on various attributes. Based on the results, choose the odorants that best suit the needs of the experimental question. This was the method used in the original experiment using direct presentation and OCDM (see original article for details on chosen odorants)15.</li>
			<li>Alternatively, the choose the odorants based on a pre-existing database containing ratings22. Exercise caution with this method due to large potential cultural differences in odor perception23.</li>
			<li>Lastly, in certain cases, choose stimuli based on the subject&#8217;s idiosyncratic odor ratings24.</li>
		</ol>
		</li>
		<li>Choose the odorous material that is best suited for the olfactometer being used.
		<ol>
			<li>Decide on liquid or solid material&#8212;some olfactometers only allow for the use of liquid material, while others allow for the use of either liquid or solid material.</li>
			<li>Obtain odorous material from a commercial supplier, either from companies that specifically produce odorants and chemicals for research purposes, or from companies that produce products (e.g., essential oils, perfumes, scented candles, etc.) for use by the general public.</li>
			<li>Alternatively, olfactometer permitting, collect odorous material from surrounding sources (e.g., pine needles, sweaty t-shirts, fruits, etc.). This method may be appropriate for some research questions; exercise caution as it makes standardization (and therefore comparisons with other research projects) difficult.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The method described here, in which an olfactometer is used to directly present odorants as contextual stimuli, represents an expansion of the utility of olfactometers in olfactory memory research16,17. Specifically, this method allows for an expansion of the area of OCDM research. Previous research had shown that odors are indeed effective contextual mnemonic cues4,14, but all research to this point has ...

Retrieval is enhanced when it occurs in the same context as encoding1,2; this is termed context dependent memory (CDM). Many stimuli have been shown to be effective contextual cues for enhancing retrieval3. In line with this general research, it has been shown that information that is learned and retrieved in the presence of the same odor is remembered better than information that is retrieved in the presence of a different odor4 or information that is neither learned nor retrieved in the presence of an extra olfactory context5; this has been termed olfactory context dependent memory (OCDM).
The use of odors as contextual mnemonic cues provides researchers with new opportunities to investigate various questions related to both CDM and olfactory processing. Indeed, the relationship between olfaction and memory has been shown to be unique6,7,8. Memories for odors seem to be more resistant to forgetting than memory for other stimuli6 (though this is not always found9). Furthermore, autobiographical memories that are cued by odors are experienced differently than autobiographical memories cued by other stimuli7.
There are other interesting differences between olfaction and the &#34;major&#34; (i.e., visual and auditory) senses. For example, when humans see or hear stimuli, we are frequently able to explicitly identify them and we often consciously adjust our behavior in line with the information they present. However, we are frequently unable to identify odors10. Furthermore, although humans are constantly surrounded by odorous substances, we do not always have the conscious experience of perceiving an odor. Accordingly, it has been proposed that olfaction is an implicit sense, and that odors mostly influence our behavior when we do not consciously perceive them11.
If olfaction truly is an implicit sense, it poses the question as to whether odors must necessarily be outside the focus of awareness for them to act as effective contextual mnemonic cues. Some evidence would suggest this is the case, as odors have been shown to be less effective cues in paired-associates paradigms then other stimuli12,13. To this point all studies that have shown evidence of OCDM have utilized ambient presentation of odorants, whereby the odorants are presented in the background and subjects learn some other target information4,14. Often, the subjects are never explicitly told about the odors.
The focus on ambient odorant presentation up to this point has been logical, as odorants are generally present in the background of an experience, but odors are infrequently at the center of our attentional focus. That being said, ambient odorant presentation necessarily means that researchers relinquish control over some aspects that may be of interest. Chief among these aspects is time. With ambient presentation odorants must be present for a long period of time, with no control over the moment of presentation or odor perception. However, presenting odorants directly, for example with the aid of an olfactometer, allows the researcher to manipulate the time of the odorant presentation in relation to the onset time of other stimuli. Further aspects of control that are gained through direct presentation include the ability to alternate between two or more odorants during a single session, presentation to one or both nostrils, presentation of &#34;no odor&#34; trials, and even the ability to manipulate the intensity of odor/concentration of odorant. In short, ambient odorant presentation limits experimenters to between subjects or block-wise manipulations, while direct odorant presentation allows for odor manipulations on a trial by trial basis.
Here the fundamentals of a method for testing OCDM using direct presentation of odorants with the aid of an olfactometer are presented15. An olfactometer is a machine that allows for the presentation of odorants at exact points in time (for example, contemporaneously with the onset of the presentation of a visual stimulus) and for a discrete amount of time (e.g., the presentation of a puff of air for 2 s). Furthermore, the olfactometer allows for the presentation to be directed below the nostrils, directly in one nostril, or directly in both nostrils. Olfactometers have long been utilized in memory research16,17. This protocol builds on previous research and applies it to the area of OCDM research.

<table><tbody><tr><td>Olfactometer</td><td>Burghart Messtechnik</td><td>OG001</td><td>Discrete presentation olfactometer</td></tr><tr><td>Odiferous material_Onion</td><td>Burghart Messtechnik</td><td>LA-13-00236</td><td>Liquid used in olfactometer to produce onion odor</td></tr><tr><td>Odiferous material_Peach</td><td>Burghart Messtechnik</td><td>LA-13-00245</td><td>Liquid used in olfactometer to produce peach odor</td></tr><tr><td>E-Prime 2.0</td><td>Psychology Software Tools</td><td></td><td>Experiment control software</td></tr></tbody></table>

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.

One of the main purposes of the current experiment was to determine whether OCDM effects could be found when the olfactory context was presented directly, rather than ambiently. There were three main dependent variables (in line with common signal detection theory analyses26): the proportion of hits, the proportion of false alarms, and a corrected accuracy score termed d&#39;. Given the focus of interest here, the data from the two odor groups from the ori...

Watch this Scientific Journal Video about Olfactory Context Dependent Memory: Direct Presentation of Odorants at JoVE.com

Olfactory Context Dependent Memory: Direct Presentation of Odorants

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 ...

olfactory-context-dependent-memory-direct-presentation-of-odorants

Research

JoVE Journal

Behavior

6.5K Views.  Universität Hildesheim. Deze methode kan ons helpen bij het beantwoorden van belangrijke vragen met betrekking tot reukconsensorische geheugen, zoals de vraag of geuren moeten gelijktijdig worden gepresenteerd met doelen om effectieve ezelsbruggetjes signalen. Het belangrijkste voordeel van deze techniek is dat directe presentatie van geurstoffen de onderzoeker in staat stelt om meer experimentele controle uit te oefenen dan omgevingspresentatie van geurstoffen. Om de olfactometer voor te bereiden, snijd de buizen o...

Video: Olfactory Context Dependent Memory: Direct Presentation of Odorants

Simple and Computer-assisted Olfactory Testing for Mice

Olfaction is highly conserved among species and is required for reproduction and survival.
In humans, olfaction is also one of the senses that is affected with aging and is a strong predictor of neurodegenerative diseases. Thus, olfaction testing is used as a non-invasive diagnostic method to detect neurological deficits early on. In order to understand the mechanisms underlying olfactory network susceptibility, olfactory research in rodents has gained momentum in the past decade.
Here, we present a very simple, time efficient and reproducible olfactory testing method of innate odor perception and sensitivity in mice without the need of any prior food or water restriction. The tests are performed in a familiar environment to the mice, require only the scents and a 2 min session of odorant exposure. The analysis is performed, post-hoc, using computer-assisted commands on ImageJ and can be, therefore, carried out from start to end by one researcher.
This protocol does not require any special hardware or setup and is indicated for any laboratory interested in testing olfactory perception and sensitivity.

We present a simple and unbiased olfactory test in mice. With this protocol olfactory discrimination, preference, avoidance and sensitivity to a novel odor as compared to water can be assessed in single behavioral sessions. This method is indicated for a single experimenter and analysis is based on computer-assisted video processing.

Olfaction is highly conserved among species and is required for reproduction and survival.
In humans, olfaction is also one of the senses that is affected ...

Testing for Metacognitive Responding Using an Odor-based Delayed Match-to-Sample Test in Rats

Metamemory involves the cognitive ability to assess the strength of one&#39;s memories. To explore the possibility of metamemory in non-human animals, numerous behavioral tasks have been created, many of which utilize an option to decline memory tests. To assess metamemory in rats, we utilized this decline-test option paradigm by adapting previous visual delayed-match-to-sample tests (DMTS)1,2 developed for primate species to an odor-based test suitable for rodents. First,&#160;rats are given a sample to remember by digging in a cup of scented sand. After a delay, the rat is presented with four distinctly scented cups, one of which contains the identical scent experienced during the sample; if this matching cup is selected, then the rat obtains a preferred, larger reward. Selection of any of the other three non-matching sand-filled scented cups results in no reward. Retention intervals are individually titrated such that subjects perform between 40 and 70% correct, therefore ensuring rats sometimes remember and sometimes forget the sample. Here, the operational definition of metamemory is the ability to distinguish between the presence and absence of memory through behavioral responding. Towards this end, on two-thirds of trials, a decline option is presented in addition to the four choice cups (choice trials). If the decline-test option- an unscented colored sand cup, is selected, the subject receives a smaller less-preferred reward and avoids the memory test. On the remaining third of trials, the decline-test option is not available (forced trials), causing subjects to guess the correct cup when the sample is forgotten. On choice tests, subjects that know when they remember should select the decline option when memory is weak rather than take the test and choose incorrectly. Therefore, significantly higher performance on chosen tests as compared to forced memory tests is indicative of the adaptive use of the decline-test response and metacognitive responding.

This protocol describes a method for investigating the possibility of metamemory, or memory awareness, in rodents. The odor-based delayed-matching-to-sample paradigm is a novel, ecologically-relevant behavioral test useful for determining the extent to which rodents can adaptively respond based on cognitively monitoring the strength of their memory states.

Metamemory involves the cognitive ability to assess the strength of one&#39;s memories. To explore the possibility of metamemory in non-human animals, ...

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 ...

Neuroscience

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 ...

An Inescapable Cat Odor Exposure Protocol for Studying Innate and Contextual Threat Conditioning in Rats

Animals respond to threatening situations by exhibiting a number of defensive behaviors, including avoidance, freezing, and risk assessment. An animal model with an ethological approach offers a deeper insight into the biological mechanisms underlying threat responses. This paper describes a methodology to measure defensive behaviors toward both innate and learned aversive stimuli in rats. Animals were individually exposed to predator odor in an inescapable chamber to elicit a measurable, sustained, defensive state. The experimental design involved placing a rat in a familiar chamber for 10 min followed by exposure to cat odor for another 10 min in the same context. The next day, the rats were re-exposed for 10 min to the same context chamber where cat odor exposure occurred. Sessions were video-recorded and defensive behaviors were assessed on both days. 
The behavioral test was coupled with reversible functional inactivation and c-Fos immunohistochemistry techniques to determine the role of the interoceptive cortex in threat responses. Rats exposed to cat odor on the first day and re-exposed to the context chamber on the second day displayed higher levels of defensive behaviors, and that cat odor elicited a robust increase in the neural activity of the interoceptive cortex. Moreover, muscimol inactivation of the interoceptive cortex reduced the expression of defensive behaviors in response to cat odor and impaired contextual threat memory. These results show that this behavioral assay is a useful tool for studying neural mechanisms of defensive behaviors and may offer insight into mechanisms that mediate fear in humans and its related disorders.

Cat skin/fur odor exposure provides a reliable method to investigate neural circuits and mechanisms of defensive responses in rodents and may offer insight into mechanisms that mediate fear in humans. Here, we describe a protocol for investigating the role of the interoceptive cortex in threat responses in rats.

Animals respond to threatening situations by exhibiting a number of defensive behaviors, including avoidance, freezing, and risk assessment. An animal ...

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.

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 ...

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. ...

Olfactory Experience-Induced Synaptic Remodeling in Drosophila's Juvenile Brain

Experience-Dependent Remodeling of Juvenile Brain Olfactory Sensory Neuron Synaptic Connectivity in an Early-Life Critical Period

Early-life olfactory sensory experience induces dramatic synaptic glomeruli remodeling in the Drosophila juvenile brain, which is experientially dose-dependent, temporally restricted, and transiently reversible only in a short, well-defined critical period. The directionality of brain circuit synaptic connectivity remodeling is determined by the specific odorant acting on the respondent receptor class of olfactory sensory neurons. In general, each neuron class expresses only a single odorant receptor and innervates a single olfactory synaptic glomerulus. In the Drosophila genetic model, the full array of olfactory glomeruli has been precisely mapped by odorant responsiveness and behavioral output. Ethyl butyrate (EB) odorant activates Or42a receptor neurons innervating the VM7 glomerulus. During the early-life critical period, EB experience drives dose-dependent synapse elimination in the Or42a olfactory sensory neurons. Timed periods of dosed EB odorant exposure allow investigation of experience-dependent circuit connectivity pruning in juvenile brain. Confocal microscopy imaging of antennal lobe synaptic glomeruli is done with Or42a receptor-driven transgenic markers that provide quantification of synapse number and innervation volume. The sophisticated Drosophila genetic toolkit enables the systematic dissection of the cellular and molecular mechanisms mediating brain circuit remodeling.

Author Spotlight: Exploring Glial Influence in Experience-Dependent Synaptic Pruning During Critical Periods

We describe here methods for inducing and analyzing olfactory experience-dependent remodeling of antennal lobe synaptic glomeruli in the Drosophila juvenile brain.

Early-life olfactory sensory experience induces dramatic synaptic glomeruli remodeling in the Drosophila juvenile brain, which is experientially ...

Olfaction

The sense of smell is achieved through the activities of the olfactory system. It starts when an airborne odorant enters the nasal cavity and reaches olfactory epithelium (OE). The OE is protected by a thin layer of mucus, which also serves the purpose of dissolving more complex compounds into simpler chemical odorants. The size of the OE and the density of sensory neurons varies among species; in humans, the OE is only about 9-10 cm2.
The olfactory receptors are embedded in the cilia of the olfactory sensory neurons. Each neuron expresses only one type of olfactory receptor. However, each type of olfactory receptor is broadly tuned and can bind to multiple different odorants. For example, if receptor A binds to odorants 1 and 2, receptor B may bind to odorants 2 and 3, while receptor C binds to odorants 1 and 3. Thus, the detection and identification of an odor depend on the combination of olfactory receptors that recognize the odor; this is called combinatorial diversity.
Olfactory sensory neurons are bipolar cells with a single long axon that sends olfactory information up to the olfactory bulb (OB). The OB is a part of the brain that is separated from the nasal cavity by the cribriform plate. Because of this convenient proximity between the nose and brain, the development of nasal drug applications is widely studied, especially in cases where direct access to the central nervous system is preferred.
Within the OB, axons from sensory neurons terminate in a specialized area called a glomerulus. Sensory neurons with the same olfactory receptor type send their axons to the same one or two glomeruli. As a result, there can be thousands of axons from similar sensory neurons converging within a single glomerulus. All of that sensory information is passed on to only 20-50 mitral and tufted cells per glomeruli, so there is a large convergence of information. Periglomerular and granular cells are inhibitory interneurons that mediate cross-talk between mitral/tufted cells before the olfactory information is sent to the cortex.
From the OB, the mitral/tufted cells project information to the olfactory cortex. The olfactory cortex is a complex of several cortical areas that process olfactory information. One olfactory area, the cortical amygdala, influences emotional responses to smell. The orbitofrontal cortex is involved in the identification of odors and the reward value of odors and tastes. The entorhinal cortex, another olfactory cortical area, projects to the hippocampus, which is implicated in olfactory memory.
The ability to detect and identify odors involves higher-order cortical areas. Such high-level integration may be linked to the impaired olfactory functioning observed in many neurodegenerative disorders, such as Parkinson&rsquo;s and Alzheimer&rsquo;s diseases. The reduced ability to smell&mdash;hyposmia&mdash;is an early symptom of both disorders.

Olfaction: Nasal Cavity and Perception of Smell

The sense of smell is achieved through the activities of the olfactory system. It starts when an airborne odorant enters the nasal cavity and reaches ...

Olfactory Context Dependent Memory: Direct Presentation of Odorants

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