This work was funded in part by the Office of Naval Research and the National Institute of Justice (2006-DN-BX-K027). The authors wish to thank the many &#8220;Furton Group&#8221; students that have participated in this project, as well as collaborators from the U.S. Naval Research Laboratory and the Naval Surface Warfare Center (Indian Head EOD Technology Division). Finally, the authors thank Peter Nunez of U.S. K-9 Academy, Tony Guzman of Metro-Dade K9 Services, and Miami-Dade area law enforcement canine teams.

1. Assembly of COMPS (Figure 1)
<ol>
	<li>For neat (liquid) compound on a substrate (Figure 1A)
	<ol>
		<li>To impregnate the substrate with odorant, use a calibrated pipette to add 5 &#181;L of neat compound to a 2 x 2 inch cotton gauze pad or other substrate of choice (see Table of Materials).</li>
		<li>Fold the gauze pad in half and place this (or an alternative substrate material) into a 2 x 3 inch low-density polyethylene permeable bag. The suggested bag thicknesses are between 1 MIL, for the fastest permeation rate, to 8 MIL, for a slower permeation rate. 
		NOTE: Variations in absorbent materials, permeable bag size, polymer chemistry, and thickness can be used, but these changes affect the permeation rate of the odorants (see further discussion in the Results Section).</li>
		<li>Immediately seal the polymer bag closed with a heat sealer, eliminating as much air from within the bags as possible.</li>
		<li>Store the bag in an outer nonpermeable bag, or if it will be used immediately, place it in a clean weigh boat in a fume hood (Figure 1B).</li>
	</ol>
	</li>
	<li>For solid material, no substrate necessary (Figure 1C)
	<ol>
		<li>Weigh the desired amount of solid material, which may be a pure compound or actual target material, and place in a 2 x 3 inch low-density polyethylene (LDPE) permeable bag. Again, the suggested bag thickness ranges from 1 MIL to 8 MIL.</li>
		<li>Immediately heat-seal the polymer bag closed, eliminating as much air from within the bag as possible, and store or set aside in a weigh boat.</li>
	</ol>
	</li>
</ol>
2. Gravimetric analysis to determine COMPS permeation rate
NOTE: A constant ambient temperature is important for accurate and reproducible measurements, both gravimetric and headspace. A constant temperature must be maintained during all testing. It is recommended to carry out all analytical measurements at desired temperatures during testing.
<ol>
	<li>To determine the permeation rate of the odorants through the permeable bag, place a newly made COMPS in a weigh boat inside of a fume hood.</li>
	<li>Place a clean, separate weigh boat on an analytical balance, and zero the balance.</li>
	<li>Remove the COMPS from the fume hood and place on the balance. Record the mass and immediately return to the fume hood.</li>
	<li>Continue recording the mass of the COMPS over regular time increments until the mass of the COMPS no longer changes (&#177;5%). At this point the odor from the COMPS is depleted.</li>
	<li>As a negative control, create an empty COMPS consisting of just the substrate material without odorants sealed into the permeable bag. Treat this negative control in the same manner as the COMPS with odorant to ensure minimal fluctuations in mass over time.</li>
	<li>Calculate the permeation rate from the COMPS.
	<ol>
		<li>Plot the mass of the COMPS versus time on an X-Y plot in appropriate statistical analysis software.</li>
		<li>Fit a linear trendline to just the linear portion of the graph and display an equation on a chart. The trendline should NOT be set to include the origin. The slope of the line (i.e., m in y = m+ b) is the permeation rate in mass per unit time.</li>
	</ol>
	</li>
</ol>
3. Headspace analysis by solid phase microextraction with gas chromatography/mass spectrometry (SPME-GC/MS) (optional)
<ol>
	<li>Prepare a fresh COMPS following the instructions above and allow it to equilibrate in an open weigh boat inside of a fume hood for 30 min.</li>
	<li>Remove the COMPS from the weigh boat, place it into a 1 pint epoxy-lined metal sample container without a lid, and put it into a 1 gallon epoxy-lined metal container. The containers should be kept in a fume hood for the duration of the experiment.</li>
	<li>Allow at least 30 min for equilibration in the container prior to sampling.</li>
	<li>For sampling after equilibration, place a lid with a previously drilled 1 cm hole atop the outer container. Insert an appropriate SPME fiber through the hole on the lid to extract the analyte of interest. When the SPME fiber is not used, cover the hole with paraffin film or the like. The extraction time and fiber coating will be dependent on the type and amount of analyte vapor present, as well as the size of the sampling vessel and environmental conditions15.</li>
	<li>Remove the SPME fiber after the allotted extraction time and place in the heated inlet of a GC/MS for thermal desorption and analysis.
	<ol>
		<li>Run the GC/MS method appropriate for the compound used in the COMPS16.</li>
	</ol>
	</li>
	<li>For quantitation, compare the resulting peak area to an external calibration curve16 and/or internal standard17 as appropriate for the method and experimental design. 
	NOTE: 1) In this example, epoxy-lined metal sample containers were used, but other types of containers would also be suitable. To directly compare the odor availability to field olfactory evaluations, it would be best to use the same container, cleaned between each test, for both experiments; 2) For reproducible results, all aspects of the sampling procedure should be maintained in all replicate experiments, including but not limited to equilibration time, SPME extraction time, container type and size, and environmental conditions (i.e., temperature and humidity).</li>
</ol>
4. COMPS storage
<ol>
	<li>Place a single COMPS in a metalized barrier bag (3.5 x 4.5 inch) and heat-seal to close, removing as much air as possible from the bag prior to sealing (Figure 1B).</li>
	<li>Store in cool ambient or refrigerated conditions but not below or close to freezing to avoid the formation of condensation as the COMPS thaws.</li>
	<li>If testing multiple odorants or odorant delivery rates in a single experiment, secondary containment is recommended to eliminate any possible cross contamination during transportation and storage.
	<ol>
		<li>Place replicate multiple barrier bags each containing individual COMPS of the same analyte and permeation rate in an outer, larger metalized bag or glass jar for storage and transport.</li>
	</ol>
	</li>
</ol>
5. Field olfactory testing
NOTE: Olfactory testing can be carried out in many different ways depending on the animal being tested, the goal of the experiment, and the environmental conditions. The protocol below describes one such manner of testing. All animal testing should first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC).
<ol>
	<li>First, create blank or negative control COMPS as described above. Make enough so every container in the testing scenario will contain spare COMPS (2&#8211;3 depending on the number of animals involved in the experiment). Package all blank COMPS together in secondary containment (i.e., a larger metalized bag or glass jar with sealing lid).</li>
	<li>Create fresh COMPS as needed for the intended field test protocol. Eliminate all possible sources of contamination between the COMPS and the metalized bag. This can be accomplished by regular changing of gloves and cleaning of the laboratory work surface.</li>
	<li>Store the COMPS for at least 1 day prior to use to allow for equilibration. Store any replicates in the same secondary container. However, different COMPS should be in separate secondary containers.</li>
	<li>To set up a basic canine olfactory test, lay out multiple lines of at least five identical containers (e.g., metal cans, boxes), with the number of lines dependent on the number of variables being tested.
	<ol>
		<li>Set up the trial so that each line contains one container with the target COMPS and four with blank COMPS. Positive control lines, prepared in the same manner but with a known target odor, may be used as appropriate for the experiment, training, or testing scenario. An additional negative control or blank line should contain five blank COMPS and no targets. Order this negative control line, positive control line (if using), and testing lines at random, and change order using a random number generator for each canine olfactory test as practical for the testing scenario.
		<ol>
			<li>Include one distractor odorant/material per line as well.</li>
			<li>Randomize the order and location of the target and distractor odorants in each line for each canine being tested using a random number generator.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>To prepare the containers, remove COMPS from the secondary and outer containers, placing only the permeable bag in the trial container.
	<ol>
		<li>Allow the COMPS to equilibrate in the container for a minimum of 30 min prior to testing.</li>
		<li>Repeat steps for each COMPS being used in the test, starting with blanks, followed by positive controls (if using), and then testing odorants, changing gloves each time. 
		NOTE: Detailed examples of canine testing scenarios can be found in Simon et al.4 or Macias et al.8.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Buck, L., Axel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+multigene+family+may+encode+odorant+receptors:+A+molecular+basis+for+odor+recognition.">A novel multigene family may encode odorant receptors: A molecular basis for odor recognition.</a> Cell. 65, 175-187 (1991).</li><li>Furton, K. G., Myers, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scientific+foundation+and+efficacy+of+the+use+of+canines+as+chemical+detectors+for+explosives.">Scientific foundation and efficacy of the use of canines as chemical detectors for explosives.</a> Talanta. 54, 487-500 (2001).</li><li>Leitch, O., Anderson, A., Kirkbride, K., Lennard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+organisms+as+volatile+compound+detectors:+A+review.">Biological organisms as volatile compound detectors: A review.</a> Forensic Science International. 232, 92-103 (2013).</li><li>Simon, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+controlled+odor+delivery+in+canine+olfactory+testing.">Method for controlled odor delivery in canine olfactory testing.</a> Chemical Senses. 44 (6), 399-408 (2019).</li><li>Hallowell, L. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+hidden+explosives:+New+challenges+and+progress+(1998-2009).">Detection of hidden explosives: New challenges and progress (1998-2009).</a> Forensic Investigation of Explosives. 2nd Ed. , 53-77 (2012).</li><li>Papet, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Narcotic+and+explosive+odors:+Volatile+organic+compounds+as+training+aids+for+olfactory+detection.">Narcotic and explosive odors: Volatile organic compounds as training aids for olfactory detection.</a> Canine Olfaction Science and Law. , 265-278 (2016).</li><li>Furton, K., Harper, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+Odor+Mimic+Permeation+System.">Controlled Odor Mimic Permeation System.</a> US Patent. , (2017).</li><li>Macias, M. S., Guerra-Diaz, P., Almirall, J. R., Furton, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+piperonal+emitted+from+polymer+controlled+odor+mimic+permeation+systems+utilizing+canis+familiaris+and+solid+phase+microextract-ion+mobility+spectrometry.">Detection of piperonal emitted from polymer controlled odor mimic permeation systems utilizing canis familiaris and solid phase microextract-ion mobility spectrometry.</a> Forensic Science International. 195, 132-138 (2010).</li><li>Harper, R., Almirall, J., Furton, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+dominant+odor+chemicals+emanating+from+explosives+for+use+in+developing+optimal+training+aid+combinations+and+mimics+for+canine+detection.">Identification of dominant odor chemicals emanating from explosives for use in developing optimal training aid combinations and mimics for canine detection.</a> Talanta. 67, 313-327 (2005).</li><li>Francis, V. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+identification+of+volatile+organic+compounds+from+synthetic+cathinone+derivatives+for+the+development+of+odor+mimic+training+aids.">The identification of volatile organic compounds from synthetic cathinone derivatives for the development of odor mimic training aids.</a> Florida International University. , (2017).</li><li>Huertas-Rivera, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+active+odors+from+illicit+substances+for+the+development+of+optimal+canine+training+aids.">Identification of the active odors from illicit substances for the development of optimal canine training aids.</a> Florida International University. , (2016).</li><li>DeGreeff, L. E., Furton, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collection+and+identification+of+human+remains+volatiles+by+non-contact,+dynamic+airflow+sampling+and+SPME-GC/MS+using+various+sorbent+materials.">Collection and identification of human remains volatiles by non-contact, dynamic airflow sampling and SPME-GC/MS using various sorbent materials.</a> Analytical and Bioanalytical Chemistry. 401, 1295-1307 (2011).</li><li>DeGreeff, L. E., Curran, A. M., Furton, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+selected+sorbent+materials+for+the+collection+of+volatile+organic+compounds+related+to+human+scent+using+non-contact+sampling+mode.">Evaluation of selected sorbent materials for the collection of volatile organic compounds related to human scent using non-contact sampling mode.</a> Forensic Science International. 209 (1-3), 133-142 (2011).</li><li>Simon, A. G., Mills, D. K., Furton, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+and+canine+analysis+as+complimentary+techniques+for+the+identification+of+active+odors+of+the+invasive+fungs,+Raffaelea+lauicola.">Chemical and canine analysis as complimentary techniques for the identification of active odors of the invasive fungs, Raffaelea lauicola.</a> Talanta. 168, 320-328 (2017).</li><li>Penton, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+development+with+solid+phase+microextraction.">Method development with solid phase microextraction.</a> Solid Phase Microextraction: A Practical Guide. , 27-58 (1999).</li><li>Robards, K., Haddad, P. R., Jackson, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles and Practice of Modern Chromatographic Methods. , (2004).</li><li>MacCrehan, W., Moore, S., Schantz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+headspace+component+vapor-time+profiles+by+solid-phase+microextraction+with+external+sampling+of+an+internal+standard.">Evaluating headspace component vapor-time profiles by solid-phase microextraction with external sampling of an internal standard.</a> Analytical Chemistry. 83, 8560-8565 (2011).</li><li>Macias, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Development of an Optimized System of Narcotic and Explosive Contraband Mimics for Calibration and Training of Biological Detectors. , (2009).</li><li>Simon, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Detection of an Invasive Pathogen through Chemical and Biological Means for the Protection of Commercial Crops. , (2017).</li></ol>

No conflicts of interest to report.

Controlled Odor Mimic Permeation Systems (COMPS) are easily created by sealing an odorant of interest into a permeable bag. This may be done by pipetting a neat liquid compound onto an absorbent material and then placing the absorbent material into the bag; by placing a pure, solid compound directly into the bag4, as was done in the case of piperonal8; or by placing the target material containing multiple or unknown odorants into a permeable bag, as was done with fungus-inf...

Olfaction is a crucial, yet often overlooked, sensing mechanism used by most animals. For many it is the main mechanism for locating food, finding a mate, or sensing danger1. Furthermore, the olfactory capabilities some animals, most notably canines, are regularly exploited by humans for the detection of contraband (e.g., narcotics or explosives), or other objects of interest, such as missing persons, invasive species, or diseases2,3. For canine detection research or other olfaction research topics, investigators often study the process of olfaction and the strengths and limitations of the olfactory system. As such, it is generally desirable to control the release of an odorant vapor into the environment to reproducibly deliver known quantities of odorant during testing. Failure to account for variations in odor availability due to factors such as vapor pressure or environmental effects often complicates data interpretation and applicability4. It is similarly desirable to provide an established quantity of odor during training scenarios for detection canines. For example, studies by Hallowell et al.5 and by Papet6 have indicated the importance of odor intensity in odor perception, and that altering the intensity of an odorant can affect how it is perceived alone or in a mixture.
In laboratory settings, the use of analytical equipment such as permeation tubes with controllable ovens, vapor generators, or olfactometers may be used to control odor delivery. However, this type of equipment is impractical for use during field testing and training scenarios4. The Controlled Odor Mimic Permeation System (COMPS) was developed as a simple, low-cost, and disposable method for controlled odor delivery requiring no external power. Therefore, they can easily be incorporated into a variety of different testing and training scenarios7. COMPS units are simply composed of an odorant of interest on an absorbent material sealed inside of a permeable polymer bag, stored in a secondary containment system. The utilization of COMPS reduces variability between tests and improves consistency during training exercises8.
Odor delivery or availability from COMPS is measured in terms of permeation rate, as determined by gravimetric analysis in terms of mass of vapor released over time. Permeation rates can be controlled by a number of factors, including the thickness of the polymer bag, its available surface area, the type of absorbent material (substrate) used, and the amount of the odorant. Permeation rate is constant for a given period of time (hours or days) depending on the odorant being used. This allows for minimal variability in odor delivery during testing or training. During storage, COMPS come to equilibrium within the impermeable outer container, resulting in an instant source of odorant vapor at a known permeation rate.
COMPS were initially designed to contain odorants associated with explosive materials and to be used as odor mimics7. As defined by Macias et al., an odor mimic simulates a material of interest, such as an explosive, by providing the dominant volatile compounds, or odorants, found in the headspace of that material without the presence of the parent material itself8. To create an odor mimic, the active odorants of the parent material must be determined. An active odorant, in this scenario, is described as a volatile compound that a trained explosive-detection canine detects, believing that there is an actual explosive material present. Having identified dominant volatile compounds in the headspace of several explosive materials, COMPS were prepared to release these individual odorants at a controlled rate for the duration of canine olfactory detection field trials and determine the active odorant associated with several explosive materials. COMPS were successfully used for this purpose7,9 and have since been used as odor mimics for further explosive detection training.
Macias et al. utilized COMPS containing piperonal, a pure chemical solid at room temperature that, in the vapor phase, has been shown to be the active odorant for MDMA (3,4-methylenedioxymethamphetamine), the psychoactive drug known as ecstasy. The researchers used varying thicknesses and surface areas of low-density polyethylene bags to adjust the permeation rate of piperonal vapor. This series of COMPS was then used to estimate piperonal detection threshold for trained narcotics-detection canines8. Conversely, in a separate study, COMPS bag thicknesses were adjusted to minimize the deviation of permeation rates between each compound in a homologous series though they possessed drastically varying vapor pressures. If a single bag thickness had been used in this study, those compounds with higher vapor pressures would have yielded much higher permeation rates. By increasing the bag thickness for the higher volatility compounds, the permeations rates were adjusted so that they were similar for all compounds4. Both studies demonstrate the utility and adaptability of the COMPS to control vapor release. Similar studies optimizing polymer bag thickness as well as absorbent material have been carried out in the creation of odor mimics for synthetic cathinones (i.e., bath salts)10, other narcotics (including heroin and marijuana11), and human odor compounds12,13. In a final example, Simon et al. investigated the active odorants associated with an invasive fungus species14. Whole pieces of infected tree bark, instead of the extracted odorants, were placed directly into the polymer bag to control release during canine olfaction testing14. COMPS can be utilized for a variety of scenarios, and the protocols discussed herein were chosen to demonstrate the diversity of this tool.

<table><tbody><tr><td>16 oz economy jars (70-450 finish)</td><td>Fillmore container</td><td>A16-08C-Case 12</td><td></td></tr><tr><td>7890A gas chromatograph / 5975 mass selective detector</td><td>Agilent</td><td></td><td></td></tr><tr><td>Analytical balance</td><td>Mettler Toledo</td><td>01-911-005</td><td></td></tr><tr><td>Ball regualr bands and dome lids</td><td>Fillmore container</td><td>J30000</td><td></td></tr><tr><td>Cotton gauze (2&quot; x 2&quot;)</td><td>Dukal</td><td></td><td></td></tr><tr><td>Disposable weighing boats</td><td>VWR</td><td>10803-148</td><td></td></tr><tr><td>Epoxy-lined sample containers, 1 gallon</td><td>TriTech Forensics</td><td>CANG-E</td><td></td></tr><tr><td>Epoxy-lined sample containers, 1 pint</td><td>TriTech Forensics</td><td>CANPT-E</td><td></td></tr><tr><td>Low density polyetheylene bag</td><td>Uline</td><td>S-5373</td><td></td></tr><tr><td>Rtx-Volatiles (30 m x 0.32 mmID) column</td><td>Restek</td><td>10901</td><td></td></tr><tr><td>Silver metalized mylar barrier bag (3.5&quot; x 4.5&quot;)</td><td>ESP Packaging</td><td>95509993779</td><td></td></tr><tr><td>Silver metalized mylar barrier bag (5&quot; x 8.5&quot; x 3&quot;)</td><td>ESP Packaging</td><td>95509993793</td><td></td></tr><tr><td>Solid phase microextration fiber assembly (PDMS/DVB/CAR)</td><td>Sigma-Aldrich</td><td>57328-U</td><td></td></tr><tr><td>Solid phase microextration holder</td><td>Sigma-Aldrich</td><td>57330-U</td><td></td></tr><tr><td>Tabletop Impulse Sealer</td><td>Uline</td><td>H-190</td><td>Heat sealer</td></tr></tbody></table>

controlled odor mimic permeation systems for olfactory training and field testing

The Controlled Odor Mimic Permeation System (COMPS) was developed to provide a convenient field testing method of odor delivery at controlled and reproducible rates. COMPS are composed of an odorant of interest on an absorbent material sealed inside of a permeable polymer bag. The permeable layer allows for a constant release of the odorant over a given amount of time. The permeable bag is further stored in a secondary, impermeable bag. The double-containment procedure allows for equilibration of the odorant from the permeable bag but within the impermeable outer layer, resulting in an instant and reproducible source of odorant vapor upon removal from the outer packaging. COMPS are used in both olfactory testing for experimental scenarios and for olfactory detection training, such as with detection canines. COMPS can be used to contain a wide range of odorants (e.g., narcotics powders) and provide a controlled release of the associated odorants. Odor availability from COMPS is expressed in terms of permeation rate (i.e., the rate of the odorant vapor released from a COMPS per unit time) and is typically measured by gravimetric means. The permeation rate for a given mass or volume of odorant can be adjusted as needed by varying the bag thickness, surface area, and/or polymer type. The available odor concentration from a COMPS can also be measured by headspace analysis techniques such as solid phase microextraction with gas chromatography/mass spectrometry (SPME-GC/MS).

The primary objective of using COMPS in olfactory testing/training is to control the release of the chosen odorants and deliver a controlled amount of the odorant over the duration of the test or training session. Odorant release is measured by gravimetric analysis in terms of mass loss per unit time. Figure 2 gives an example of gravimetric results from the permeation of three identical COMPS prepared from 5 &#181;L of pentanoic acid on cotton gauze through a 3 MIL LDPE bag. A line of regre...

Watch this Scientific Journal Video about Controlled Odor Mimic Permeation Systems for Olfactory Training and Field Testing at JoVE.com

Controlled Odor Mimic Permeation Systems for Olfactory Training and Field Testing

The Controlled Odor Mimic Permeation System is a simple, field-portable, low-cost method of odor delivery for olfactory testing and training. It is constructed of an odorant retained on an adsorbent material and contained inside of a permeable polymer bag allowing controlled release of the odorant vapor over time.

The Controlled Odor Mimic Permeation System (COMPS) was developed to provide a convenient field testing method of odor delivery at controlled and ...

controlled-odor-mimic-permeation-systems-for-olfactory-training-field

Research

JoVE Journal

Chemistry

4.5K Views.  US Naval Research Laboratory. Olfactorische onderzoek wordt beperkt door het gebrek aan methodologie voor het leveren van bekende en reproduceerbare geuren tijdens operationele testen of training. COMPS biedt een handige fieldable methode voor gecontroleerde geurlevering. COMPS kan een breed scala aan geurstoffen bevatten en de leveringspercentages kunnen worden gevarieerd door de dikte van de zak, het oppervlak en/of het polymeertype aan te passen.Om een substraat met geurstof te impregneren, gebruik dan een gekal...

Video: Controlled Odor Mimic Permeation Systems for Olfactory Training and Field Testing

Testing for Odor Discrimination and Habituation in Mice

This video demonstrates a technique to establish the presence of a normally functioning olfactory system in a mouse. The test helps determine whether the mouse can discriminate between non-social odors and social odors, whether the mouse habituates to a repeatedly presented odor, and whether the mouse demonstrates dishabituation when presented with a novel odor. Since many social behavior tests measure the experimental animal&#8217;s response to a familiar or novel mouse, false positives can be avoided by establishing that the animals can detect and discriminate between social odors. There are similar considerations in learning tests such as fear conditioning that use odor to create a novel environment or olfactory cues as an associative stimulus. Deficits in the olfactory system would impair the ability to distinguish between contexts and to form an association with an olfactory cue during fear conditioning. 
In the odor habitation/dishabituation test, the mouse is repeatedly presented with several odors. Each odor is presented three times for two minutes. The investigator records the sniffing time directed towards the odor as the measurement of olfactory responsiveness. A typical mouse shows a decrease in response to the odor over repeated presentations (habituation). The experimenter then presents a novel odor that elicits increased sniffing towards the new odor (dishabituation). After repeated presentation of the novel odor the animal again shows habituation. This protocol involves the presentation of water, two or more non-social odors, and two social odors. In addition to reducing experimental confounds, this test can provide information on the function of the olfactory systems of new knockout, knock-in, and conditional knockout mouse lines.

This manuscript describes a protocol to examine the olfactory system of rodents. The olfactory habituation/dishabituation test will allow investigators to determine whether a mouse habituates to a repeatedly presented odor and whether the mouse demonstrates dishabituation when presented a novel odor.

This video demonstrates a technique to establish the presence of a normally functioning olfactory system in a mouse. The test helps determine whether the ...

Behavior

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 ...

Olfactory Behavioral Testing in the Adult Mouse

The rodent olfactory system is of increasing interest to scientists, studied, in part, in systems biology because of its stereotyped, yet accessible circuitry. In addition, this area's unique ability to generate new neurons throughout an organism's lifetime makes it an attractive system for developmental and regenerative biologists alike. Such interest necessitates a means for a quick, yet reliable assessment of olfactory function. Many tests of olfactory ability are complex, variable or not specifically designed for mice. Also, some tests are sensitive to memory deficits as well as defects in olfactory abilities, confounding obtained results.
Here, we describe a simple battery of tests designed to identify defects in olfactory sensitivity and preference. First, an initial general health assessment allows for the identification of animals suitable for further testing. Second, mice are exposed to various dilutions of scents to ascertain whether there is a threshold difference. Third, mice are presented with various scents, both attractive and aversive, that allow for the assessment of olfactory preference. These simple studies should make the initial characterization of olfactory behavior accessible for labs of varied resources and expertise.

Fundamental, yet unique properties of the rodent olfactory system have led to its increasing study among biologists. A relatively simple assessment of its function is then also needed. Here we describe sensitive tests for the characterization of mouse olfactory sensitivity and preference.

The rodent olfactory system is of increasing interest to scientists, studied, in part, in systems biology because of its stereotyped, yet accessible ...

Biology

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 ...

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 ...

A Proboscis Extension Response Protocol for Investigating Behavioral Plasticity in Insects: Application to Basic, Biomedical, and Agricultural Research

Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g., food, mates, threats). There are several behavioral mechanisms through which an insect learns salient associations and relates them to these events. It is important to understand this behavioral plasticity for programs aimed toward assisting insects that are beneficial for agriculture. This understanding can also be used for discovering solutions to biomedical and agricultural problems created by insects that act as disease vectors and pests. The Proboscis Extension Response (PER) conditioning protocol was developed for honey bees (Apis mellifera) over 50 years ago to study how they perceive and learn about floral odors, which signal the nectar and pollen resources a colony needs for survival. The PER procedure provides a robust and easy-to-employ framework for studying several different ecologically relevant mechanisms of behavioral plasticity. It is easily adaptable for use with several other insect species and other behavioral reflexes. These protocols can be readily employed in conjunction with various means for monitoring neural activity in the CNS via electrophysiology or bioimaging, or for manipulating targeted neuromodulatory pathways. It is a robust assay for rapidly detecting sub-lethal effects on behavior caused by environmental stressors, toxins or pesticides.
We show how the PER protocol is straightforward to implement using two procedures. One is suitable as a laboratory exercise for students or for quick assays of the effect of an experimental treatment. The other provides more thorough control of variables, which is important for studies of behavioral conditioning. We show how several measures for the behavioral response ranging from binary yes/no to more continuous variable like latency and duration of proboscis extension can be used to test hypotheses. And, we discuss some pitfalls that researchers commonly encounter when they use the procedure for the first time.

The Proboscis Extension Response (PER) conditioning protocol, developed for the honey bee (Apis mellifera), provides an ecologically-relevant and easily quantifiable means for studying several different mechanisms of learning in many insect species.

Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g., food, mates, threats). ...

Neuroscience

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 ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis 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 ...

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an excellent model system for such investigations due to its complex behaviors, powerful genetic techniques, and compact nervous system. Laboratory behavioral assays have long been used with Drosophila to simulate properties of the natural environment and study the neural mechanisms underlying the corresponding behaviors (e.g. phototaxis, chemotaxis, sensory learning and memory)1-3. With the recent availability of large collections of transgenic Drosophila lines that label specific neural subsets, behavioral assays have taken on a prominent role to link neurons with behaviors4-11. Versatile and reproducible paradigms, together with the underlying computational routines for data analysis, are indispensable for rapid tests of candidate fly lines with various genotypes. Particularly useful are setups that are flexible in the number of animals tested, duration of experiments and nature of presented stimuli. The assay of choice should also generate reproducible data that is easy to acquire and analyze. Here, we present a detailed description of a system and protocol for assaying behavioral responses of Drosophila flies in a large four-field arena. The setup is used here to assay responses of flies to a single olfactory stimulus; however, the same setup may be modified to test multiple olfactory, visual or optogenetic stimuli, or a combination of these. The olfactometer setup records the activity of fly populations responding to odors, and computational analytical methods are applied to quantify fly behaviors. The collected data are analyzed to get a quick read-out of an experimental run, which is essential for efficient data collection and the optimization of experimental conditions.

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

We describe here a behavioral setup and data analysis method for assaying olfactory responses of up to 100 vinegar flies (Drosophila melanogaster). This system may be used with single or multiple olfactory stimuli, and adaptable for optogenetic activation or silencing of neuronal subsets.

Controlled Odor Mimic Permeation Systems for Olfactory Training and Field Testing

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