Name: sampling and analysis of animal scent signals
Uploaded: 2021-02-13T12:30:00.000+00:00
Duration: 14 min 59 s
Description: Watch this Scientific Journal Video about Sampling and Analysis of Animal Scent Signals at JoVE.com

We thank Keith Holding for his assistance with chemical analyses at the Rosalind Franklin Science Center, Wolverhampton, and Ben Mantle for the production of the video. We are also grateful to Prof. Gloriano Moneti, Dr. Giuseppe Pieraccini and the members of the University of Florence&#39;s Mass Spectrometry Center, Florence, and to Prof. Luca Calamai and Dr. Marco Michelozzi of the CNR&#39;s ARCA Lab, Florence, for their help with setting up this methodology. The research projects which included the sampling and analysis methods described in the manuscript were supported by two Marie Sk&#322;odowska-Curie Intra European Fellowships (Grant Agreement IDs: 327083, 703611), a small grant (&#39;The sensory enriched primate&#39;) from the Primate Society of Great Britain, and a small research grant (&#39;Do hunter-gatherers have a special sense of smell?&#39;) from the British Academy/The Leverhulme Trust to S.V. The lab work needed to set up this methodology also received funding from the Faculty of Science and Engineering&#39;s Annual Funding Competition (Wolverhampton) to S.V.

1. Sample collection
<ol>
	<li>Sample odors that are one of the following:
	<ol>
		<li>Collect spontaneously released by habituated study subjects (e.g., zoo primates) via scent-marking on sterile filter paper (e.g., scent-gland odor secretions) or directly into vials (e.g., urine).</li>
		<li>Collect by rubbing sterile cotton swabs after training study subjects by using positive reinforcement training.</li>
		<li>Collect by rubbing sterile cotton swabs after sedation of study subjects.</li>
	</ol>
	</li>
	<li>Place samples into sterile 10 mL screw-capped clear glass vials, and seal with screw topped caps incorporating FTFE/silicone septa. Immediately store them at -20 &#176;C. 
	NOTE: It is vital to use clean personal protective equipment, such as nitrile gloves; change them frequently; avoid direct skin contact with samples and vials. It is preferred to use brand-new vials; however, in case of used vials, it is vital to pre-clean the vials, and then use the same protocol.</li>
	<li>Take environmental blanks every time scent-marks are collected. For example, collect the sampling media (e.g., filter paper or swab) and a headspace vial that are exposed to the environment while sampling is undertaken.</li>
</ol>
2. Sample preparation
<ol>
	<li>Prepare samples in the field by cutting with a blade an approximate 10 mm square from a scent-marked filter paper, or the head of the swab, and placing it in a 10 mL screw-topped head-space vial.</li>
	<li>After each sample has been prepared, dispose or clean the blade used to cut the sampling media using an appropriate antibacterial wipe and/or alcohol and dry thoroughly.</li>
	<li>Store all samples at -20 &#176;C. 
	NOTE: Preferred at -20 &#176;C or otherwise as low as practically possible in the field.</li>
</ol>
3. Preparation for analysis
<ol>
	<li>Remove samples from the freezer and allow to warm naturally to room temperature for at least 1 h.</li>
	<li>Set up the analytical method on the GC-MS as follows:
	<ol>
		<li>For SPME analysis conditions, follow the manufacturer&#39;s directions to condition SPME fibers before first use: fiber pre-condition (260 &#176;C for 5 min), sample incubation (40 &#176;C for 2 min), extraction time (15 min), desorption time (2 min), and fiber post-condition (260 &#176;C for 20 min).</li>
		<li>Use the following GC conditions: column (HP5-MS 30 m x 0.25 mm; 0.25 &#181;m), injector temperature (270 &#176;C), flow rate (1 mL/min), injection mode (splitless), GC oven profile (45 &#176;C for 2 min; 4 &#176;C/min to 170 &#176;C; 20 &#176;C/min to 300 &#176;C), MSD transfer line (280 &#176;C). 
		NOTE: To improve between sample retention time consistency the analytical method is retention time locked).</li>
		<li>Use the following MSD conditions: solvent delay (2.5 min) and scan range (29 to 400 amu). 
		NOTE: A range of 10 to 400 was used in previous protocols4.</li>
	</ol>
	</li>
	<li>Ensure that the purge gas supply to the fiber conditioning unit is turned on. 
	&#8203;NOTE: It is vital that the SPME assembly is correctly installed in the auto-sampler and that it is aligned to the auto-sampler trays, fiber conditioning unit and GC inlet port. Incorrect alignment could result in damage or destruction of the SPME fiber.</li>
</ol>
4. Analysis
<ol>
	<li>Place an empty headspace vial (to act as a system blank) in the first position of the GC-MS auto-sampler tray. Place the environmental blank in the second position of the auto-sampler tray. Place the samples for analysis in the subsequent positions of the auto-sample tray.</li>
	<li>Create an analytical sequence to analyze each sample within the sample tray.
	<ol>
		<li>In the MassHunter home screen, select Sequence | Load sequence.</li>
		<li>Complete the sequence table for all blanks and samples by inserting the appropriate information. Save the completed sequence table. 
		NOTE: The exact information for the sequence table will be dependent on the laboratories formatting for the table. Minimum information would normally include sample type, sample name, vial location and number, analytical method and data file location and name (allocation of a data file name that matches the sample name aids future data processing). Additional samples can be added to the sequence during the analysis.</li>
	</ol>
	</li>
	<li>Run the sequence by selecting Sequence | Run sequence.</li>
	<li>After analysis return samples to the freezer as soon as possible. 
	&#8203;NOTE: it may be possible to reanalyze samples, but it should be noted that some volatile components may have been totally extracted during the initial analysis and some compounds may have undergone thermal and bacterial decomposition at 40 &#176;C, thus the resulting chromatogram may not be thoroughly representative of the original scent-marking.</li>
</ol>
5. Data analysis
NOTE: Initial data analysis includes the integration of chromatograms to obtain retention time and peak area data together with tentative identification of peaks using ChemStation software and NIST (National Institute of Standards and Technology) mass spectral databases, version MSD F.01.01.2317. Data analysis can be carried out either manually or through a semi-automated method. If the semi-automated method is used then it is sometimes beneficial to undertake a degree of manual data analysis to verify tentative identifications.
<ol>
	<li>Open the data file by clicking on the appropriate file in the left-hand navigation bar. The total ion chromatogram (TIC) will be displayed in the top window of the data analysis screen.</li>
	<li>To integrate the TIC using the RTE integrator, select Chromatogram | Integrate.</li>
	<li>Adjust integration parameters so that peaks that are greater than 3 x baseline noise are integrated. Select Chromatogram | MS Signal Integration Parameters. In the output box adjust the minimum peak area as appropriate (1.0 produces acceptable results in our examples).</li>
	<li>To identify peaks and generate a summary report, select Export Reports | Library search results report to XLS. 
	NOTE: The spectral libraries to be searched together with the number of library matches to be displayed need to be preset within the software before a library search can be undertaken.</li>
	<li>The resulting spreadsheet report contains integration data for each peak and a tentative spectral library match to assign identity. Typically, the library quality/library match should be &#62;80 to accept the tentative identification. Save the spreadsheet.</li>
	<li>Identify a peak directly from the TIC.
	<ol>
		<li>Choose the peak of interest.</li>
		<li>If the peak is small, zoom in by drawing a box around the peak by holding the left hand mouse button down, stretch the box over the peak and release.</li>
		<li>Place the cursor line so that it is at the highest point of the peak (or just after).</li>
		<li>Double click the right hand mouse button and the mass spectrum for the peak will appear in the lower window of the data analysis screen.</li>
		<li>To search the spectral library, move the cursor anywhere in the spectral window and double click the right hand mouse button. The library search results will appear in a new window.</li>
		<li>To remove the background noise from a spectrum of interest, first double click the right hand mouse button on the peak in question. Then double click the right hand mouse button in an area with no peaks immediately in front of the peak of interest. Select Chromatogram | Subtract spectra. The subtracted spectrum will display in the lower window of the data analysis screen and will display &#39;(-)&#39; next to the SCAN data in the window header.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Wyatt, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pheromones and Animal Behavior: Chemical Signals and Signatures. , (2014).</li><li>Heymann, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neglected+sense-olfaction+in+primate+behavior,+ecology,+and+evolution.">The neglected sense-olfaction in primate behavior, ecology, and evolution.</a> American Journal of Primatology. 68 (6), 519-524 (2006).</li><li>Drea, C. M., Boulet, M., DelBarco-Trillo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+"secret"+in+secretions:+Methodological+considerations+in+deciphering+primate+olfactory+communication.">The "secret" in secretions: Methodological considerations in deciphering primate olfactory communication.</a> American Journal of Primatology. 75 (7), 621-642 (2013).</li><li>Vaglio, S., 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=Sternal+gland+scent-marking+signals+sex,+age,+rank+and+group+identity+in+captive+mandrills.">Sternal gland scent-marking signals sex, age, rank and group identity in captive mandrills.</a> Chemical Senses. 41 (2), 177-186 (2016).</li><li>Marneweck, C., J&#252;rgens, A., Shrader, 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=Dung+odours+signal+sex,+age,+territorial+and+oestrous+state+in+white+rhinos.">Dung odours signal sex, age, territorial and oestrous state in white rhinos.</a> Proceedings of the Royal Society of London B: Biological Sciences. 284 (1846), (2016).</li><li>Shear, W. A., Jones, T. H., Miras, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+possible+phylogenetic+signal+in+milliped+chemical+defenses.">A possible phylogenetic signal in milliped chemical defenses.</a> Biochemical Systematics and Ecology. 35, 838-842 (2007).</li><li>Kimura, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volatile+substances+in+feces,+urine+and+urine-marked+feces+of+feral+horses.">Volatile substances in feces, urine and urine-marked feces of feral horses.</a> Canadian Journal of Animal Science. 81 (3), 411-420 (2001).</li><li>Vaglio, S., Minicozzi, P., Bonometti, E., Mello, G., Chiarelli, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volatile+signals+during+pregnancy:+a+possible+chemical+basis+for+mother-infant+recognition.">Volatile signals during pregnancy: a possible chemical basis for mother-infant recognition.</a> Journal of Chemical Ecology. 35 (1), 131-139 (2009).</li><li>Setchell, J. M., 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=Chemical+composition+of+scent-gland+secretions+in+an+Old+World+monkey+(Mandrillus+sphinx):+influence+of+sex,+male+status,+and+individual+identity.">Chemical composition of scent-gland secretions in an Old World monkey (Mandrillus sphinx): influence of sex, male status, and individual identity.</a> Chemical Senses. 35 (3), 205-220 (2010).</li><li>Setchell, J. M., 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=Odour+signals+MHC+genotype+in+an+Old+World+monkey.">Odour signals MHC genotype in an Old World monkey.</a> Proceedings of the Royal Society of London B: Biological Sciences. 278 (1703), 274-280 (2011).</li><li>Pawliszyn, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Solid phase microextraction: theory and practice. , (1997).</li><li>Janda, E. D., Perry, K., Hankinson, E., Walker, D., Vaglio, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+differences+in+scent-marking+in+captive+red-ruffed+lemurs.">Sex differences in scent-marking in captive red-ruffed lemurs.</a> American Journal of Primatology. 81 (1), 22951 (2019).</li></ol>

The authors have nothing to disclose.

The use of control samples, both environmental controls created at the time of sample collection and system blanks, are crucial for the interpretation of the scent-mark samples. Any peaks attributed to the sampling environment or the instrumental system need to be excluded from scent-mark samples so that only the peaks of interest are included in any interpretation. These controls can also play a role in assessing and monitoring the 'health' of the instrumentation.
The protocol includes steps ...

Very little is known about the chemical changes underpinning the olfactory signals in animals1, also because of methodological challenges in recording and quantifying volatile chemical profiles of odors2. There are several potential pitfalls when working with highly complex, chemical matrices; these include when sampling and analyzing the odor samples3.
At the Rosalind Franklin Science Center, University of Wolverhampton, we are undertaking the analysis of odors and scent-marks to understand how they may be used by animals. We combine semiochemistry with behavioral ecology, endocrinology and cytology to improve our understanding of the role played by olfactory signals in animal communication.
We have developed a methodology and then analyzed odors and markings from a variety of species including several non-human primates (i.e., crowned lemurs, red-ruffed lemurs, Japanese macaques, olive baboons, chimpanzees) and other mammals (i.e., cats, cows). We have collected and analyzed a variety of samples, including urine, feces, hair and scent-gland odor secretions. These odors and scent-marks consist of complex mixtures of compounds and therefore any methodology used for their analysis need to include some form of separatory technique. As illustrated, they also occur in a range of matrices which necessitates the use of techniques to extract the components of interest.
Previous studies by Vaglio et al.4 and other authors5 used dynamic headspace extraction (DHS) with gas chromatography-mass spectrometry (GC-MS) while direct solvent extraction6 and complex solvent extractions7 have also been used. Particularly, dynamic headspace sampling involves purging the headspace with a known volume of inert gas that ultimately removes all volatile compounds with the exception of those showing a strong affinity for the sample matrix (for example, polar compounds in aqueous samples).
For the current methodology, we have adopted the technique of headspace solid-phase microextraction (HS-SPME) coupled with GC-MS. In particular, we have developed and enhanced the methodology already used by Vaglio et al. in its previous GC-MS laboratory8,9,10.
Solventless extraction techniques are very effective for analyzing small, highly volatile compounds (which otherwise can be lost easily from a sample) because these methods immobilize compounds on a stable, solid phase support. The HS-SPME uses a &#64257;ber coated with an adsorbent polymer to capture volatile compounds in the sample headspace or to extract dissolved compounds by immersion in an aqueous biological &#64258;uid11. The polymer coating does not bind the compounds strongly, therefore by heating in the injection port of the GC they can be removed. This method is more powerful than solvent extraction techniques and also more effective than DHS.
In the current approach samples are contained within glass vials. These vials are warmed to a temperature of 40 &#176;C to simulate animal body temperature in order to promote the volatile components of the scent-mark to occupy the head-space of the vial. A SPME fiber, coated with 65 &#181;m of polydimethylsiloxane/divinylbenzene (PDMS/DVB) sorbent material, is exposed to the headspace environment and volatile components from the sample are adsorbed onto the fiber. On heating the fiber in the inlet port of a GC-MS, the volatile components are desorbed from the fiber and then separated by the GC. Mass spectral fragmentation patterns are obtained for each component using the MS. By comparison of these mass spectra against mass spectral databases, it can be possible to tentatively identify the components of the scent-mark. Through the use of an auto-sampler, we are able to analyze multiple samples in batches in a consistent manner.
Given that each type of SPME fiber has a different affinity with polar chemicals, the fiber is usually chosen depending on the polarity and/or molecular weight of the target chemical compounds. In addition, the GC conditions are changed depending on the type of GC column and the characteristics of the target chemical compounds.
This technique allows the semi-quantitative analysis of the volatile components of scent-markings by enabling the separation and tentative identification of the components in the sample, followed by the analysis of peak area ratios to look for trends that could signify components of the scent-marking that may be involved in signaling.
The key strengths of this current approach are:
<ul style="list-style-type: square;">
	<li>The range of sample types that can be analyzed.</li>
	<li>No complex sample preparation or extractions are required.</li>
	<li>The ability to analyze volatile components.</li>
	<li>The ability to separate the components of a mixture.</li>
	<li>To be able to identify the components detected.</li>
	<li>The ability to provide semi-quantitative and potentially quantitative information on the components detected.</li>
</ul>

<table><tbody><tr><td>10 mL autosampler vials</td><td>Agilent</td><td>5188-5392</td><td>10 ml screwtop vials with</td></tr><tr><td>18 mm vial caps</td><td>Agilent</td><td>8010-0139</td><td>Magnetic with PTFE/silicone septa</td></tr><tr><td>Autosampler</td><td>Agilent</td><td>GC120 PAL autosampler</td><td></td></tr><tr><td>Capillary column</td><td>Agilent</td><td>HP5-MS</td><td>30 m x 0.25 mm; 0.25 &amp;micro;m</td></tr><tr><td>Data analysis software</td><td>Agilent</td><td>-</td><td>ChemStation</td></tr><tr><td>Gas Chromatograph</td><td>Agilent</td><td>7890B</td><td></td></tr><tr><td>Inlet septa</td><td>Agilent</td><td>5182-3442</td><td>Merlin microseal</td></tr><tr><td>Mass Selective Detector</td><td>Agilent</td><td>5977A</td><td></td></tr><tr><td>Reporting software</td><td>Microsoft</td><td>-</td><td>Excel</td></tr><tr><td>Spectral library</td><td>NIST</td><td>-</td><td>NIST/EPA/NIH Mass Spectral Library</td></tr><tr><td>Spectral library search program</td><td>NIST</td><td>-</td><td>MS Search v.2.2</td></tr><tr><td>Splitless Inlet liner</td><td>Agilent</td><td>5190-4048</td><td></td></tr><tr><td>SPME fibres</td><td>Agilent</td><td>SU57345U</td><td>65 &amp;micro;m PDMS/DVB fibre</td></tr></tbody></table>

sampling and analysis of animal scent signals

We have developed an effective methodology for sampling and analysis of odor signals, by using headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry, to understand how they may be used in animal communication. This technique allows the semi-quantitative analysis of the volatile components of odor secretions by enabling the separation and tentative identification of the components in the sample, followed by the analysis of peak area ratios to look for trends that could signify compounds that may be involved in signaling. The key strengths of this current approach are the range of sample types that can be analyzed; the lack of need for any complex sample preparation or extractions; the ability to separate and analyze the components of a mixture; the identification of the components detected; and the capability to provide semi-quantitative and potentially quantitative information on the components detected. The main limitation to the methodology relates to the samples themselves. Since the components of specific interest are volatile, and these could easily be lost, or their concentrations altered, it is important that the samples are stored and transported appropriately after their collection. This also means that sample storage and transport conditions are relatively costly. This method can be applied to a variety of samples (including urine, feces, hair and scent-gland odor secretions). These odors consist of complex mixtures, occurring in a range of matrices, and thus necessitate the use of techniques to separate the individual components and extract the compounds of biological interest.

Following this protocol, we tentatively identified a total of 32 volatile chemical compounds from the analysis of 14 ano-genital scent-marks spontaneously released on filter paper by red-ruffed lemurs (Varecia variegata rubra) and compared odor profiles with features of the signaler12. Naturally occurring volatile compounds, such as hydrocarbons, terpenes, terpene alcohols and ketones, were present within these profiles and included compounds that had previously been found to act as sex p...

Watch this Scientific Journal Video about Sampling and Analysis of Animal Scent Signals at JoVE.com

Sampling and Analysis of Animal Scent Signals

We have developed an effective methodology for sampling and analysis of odor signals in order to understand how they may be used in animal communication. Particularly, we use headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry to analyze the volatile components of animal odors and scent-markings.

We have developed an effective methodology for sampling and analysis of odor signals, by using headspace solid-phase microextraction coupled with gas ...

sampling-and-analysis-of-animal-scent-signals

Universidad San Sebastian

Research

JoVE Journal

Behavior

4.6K Views.  University of Wolverhampton. We have developed an effective methodology for sampling and analysis of odor signals in order to understand how they may be used in animal communication. Particularly, we use headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry to analyze the volatile components of animal odors and scent-markings.

Video: Sampling and Analysis of Animal Scent Signals

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

Biology

Eliciting and Analyzing Male Mouse Ultrasonic Vocalization (USV) Songs

Mice produce ultrasonic vocalizations (USVs) in a variety of social contexts throughout development and adulthood. These USVs are used for mother-pup retrieval1, juvenile interactions2, opposite and same sex interactions3,4,5, and territorial interactions6. For decades, the USVs have been used by investigators as proxies to study neuropsychiatric and developmental or behavioral disorders7,8,9, and more recently to understand mechanisms and evolution of vocal communication among vertebrates10. Within the sexual interactions, adult male mice produce USV songs, which have some features similar to courtship songs of songbirds11. The use of such multisyllabic repertoires can increase potential flexibility and information they carry, as they can be varied in how elements are organized and recombined, namely syntax. In this protocol a reliable method to elicit USV songs from male mice in various social contexts, such as exposure to fresh female urine, anesthetized animals, and estrus females is described. This includes conditions to induce a large amount of syllables from the mice. We reduce recording of ambient noises with inexpensive sound chambers, and present a quantification method to automatically detect, classify and analyze the USVs. The latter includes evaluation of call-rate, vocal repertoire, acoustic parameters, and syntax. Various approaches and insight on using playbacks to study an animal&#39;s preference for specific song types are described. These methods were used to describe acoustic and syntax changes across different contexts in male mice, and song preferences in female mice.

Eliciting and Analyzing Male Mouse Ultrasonic Vocalization USV Songs

Mice produce a complex multisyllabic repertoire of ultrasonic vocalizations (USVs). These USVs are widely used as readouts for neuropsychiatric disorders. This protocol describes some of the practices we learned and developed to consistently induce, collect, and analyze the acoustic features and syntax of mouse songs.

Mice produce ultrasonic vocalizations (USVs) in a variety of social contexts throughout development and adulthood. These USVs are used for mother-pup ...

Using a Comparative Species Approach to Investigate the Neurobiology of Paternal Responses

A goal of behavioral neuroscience is to identify underlying neurobiological factors that regulate specific behaviors. Using animal models to accomplish this goal, many methodological strategies require invasive techniques to manipulate the intensity of the behavior of interest (e.g., lesion methods, pharmacological manipulations, microdialysis techniques, genetically-engineered animal models). The utilization of a comparative species approach allows researchers to take advantage of naturally occurring differences in response strategies existing in closely related species. In our lab, we use two species of the Peromyscus genus that differ in paternal responses. The male California deer mouse (Peromyscus californicus) exhibits the same parental responses as the female whereas its cousin, the common deer mouse (Peromyscus maniculatus) exhibits virtually no nurturing/parental responses in the presence of pups. Of specific interest in this article is an exploration of the neurobiological factors associated with the affiliative social responses exhibited by the paternal California deer mouse. Because the behavioral neuroscience approach is multifaceted, the following key components of the study will be briefly addressed: the identification of appropriate species for this type of research; data collection for behavioral analysis; preparation and sectioning of the brains; basic steps involved in immunocytochemistry for the quantification of vasopressin-immunoreactivity; the use of neuroimaging software to quantify the brain tissue; the use of a microsequencing video analysis to score behavior and, finally, the appropriate statistical analyses to provide the most informed interpretations of the research findings.

The comparative species approach allows behavioral neuroscientists to explore various neurobiological factors associated with specific behaviors viewed as characteristic of a specific animal model. Taking advantage of naturally occurring differences in behavior between closely related species, this technique doesn&#x2019;t require invasive techniques to manipulate the expression of the behavior.

A goal of behavioral neuroscience is to identify underlying neurobiological factors that regulate specific behaviors.  Using animal models to accomplish ...

Neuroscience

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. Pheromones are volatile or non-volatile short-lived molecules2 secreted and/or contained in biological fluids3,4, such as urine, a liquid known to be a main source of pheromones3. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions5,6, hierarchical organisations3 and sexual interactions7,8 and are consequently directly correlated with the survival of a given species9,10,11. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO)10,12, a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen13,14 allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs)15. Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection16. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize17,18. Two main vomeronasal receptor families, V1Rs and V2Rs19,20,21,22, are composed respectively by 24023 and 12024 members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs)25 and formyl peptide receptors (FPRs)26,27 are also expressed in VSNs.
Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons28 TRPC2 independent transduction channels have been suggested6,29. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex.
Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line30, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy.

In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO). Here, an acute tissue slice preparation of VNO for performing calcium imaging is described. This physiological approach allows observations of subpopulations and/or individual neurons in a living tissue and is convenient for receptor-ligand identification.

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. ...

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 ...

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings (GCMR) in the Insect Antennal Lobe

All organisms inhabit a world full of sensory stimuli that determine their behavioral and physiological response to their environment. Olfaction is especially important in insects, which use their olfactory systems to respond to, and discriminate amongst, complex odor stimuli. These odors elicit behaviors that mediate processes such as reproduction and habitat selection1-3. Additionally, chemical sensing by insects mediates behaviors that are highly significant for agriculture and human health, including pollination4-6, herbivory of food crops7, and transmission of disease8,9. Identification of olfactory signals and their role in insect behavior is thus important for understanding both ecological processes and human food resources and well-being.
	To date, the identification of volatiles that drive insect behavior has been difficult and often tedious. Current techniques include gas chromatography-coupled electroantennogram recording (GC-EAG), and gas chromatography-coupled single sensillum recordings (GC-SSR)10-12. These techniques proved to be vital in the identification of bioactive compounds. We have developed a method that uses gas chromatography coupled to multi-channel electrophysiological recordings (termed 'GCMR') from neurons in the antennal lobe (AL; the insect's primary olfactory center)13,14. This state-of-the-art technique allows us to probe how odor information is represented in the insect brain. Moreover, because neural responses to odors at this level of olfactory processing are highly sensitive owing to the degree of convergence of the antenna's receptor neurons into AL neurons, AL recordings will allow the detection of active constituents of natural odors efficiently and with high sensitivity. Here we describe GCMR and give an example of its use.
	Several general steps are involved in the detection of bioactive volatiles and insect response. Volatiles first need to be collected from sources of interest (in this example we use flowers from the genus Mimulus (Phyrmaceae)) and characterized as needed using standard GC-MS techniques14-16. Insects are prepared for study using minimal dissection, after which a recording electrode is inserted into the antennal lobe and multi-channel neural recording begins. Post-processing of the neural data then reveals which particular odorants cause significant neural responses by the insect nervous system.
	Although the example we present here is specific to pollination studies, GCMR can be expanded to a wide range of study organisms and volatile sources. For instance, this method can be used in the identification of odorants attracting or repelling vector insects and crop pests. Moreover, GCMR can also be used to identify attractants for beneficial insects, such as pollinators. The technique may be expanded to non-insect subjects as well.

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings GCMR in the Insect Antennal Lobe

Olfactory cues mediate many different behaviors in insects, and are often complex mixtures comprised of tens to hundreds of volatile compounds. Using gas chromatography with multi-channel recording in the insect antennal lobe, we describe a method for the identification of bioactive compounds.

All  organisms inhabit a world full of sensory stimuli that determine their  behavioral and physiological response to their environment. Olfaction is ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

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 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 ...

Chemistry

Using Insect Electroantennogram Sensors on Autonomous Robots for Olfactory Searches

Robots designed to track chemical leaks in hazardous industrial facilities1 or explosive traces in landmine fields2 face the same problem as insects foraging for food or searching for mates3: the olfactory search is constrained by the physics of turbulent transport4. The concentration landscape of wind borne odors is discontinuous and consists of sporadically located patches. A pre-requisite to olfactory search is that intermittent odor patches are detected. Because of its high speed and sensitivity5-6, the olfactory organ of insects provides a unique opportunity for detection. Insect antennae have been used in the past to detect not only sex pheromones7 but also chemicals that are relevant to humans, e.g., volatile compounds emanating from cancer cells8 or toxic and illicit substances9-11. We describe here a protocol for using insect antennae on autonomous robots and present a proof of concept for tracking odor plumes to their source. The global response of olfactory neurons is recorded in situ in the form of electroantennograms (EAGs). Our experimental design, based on a whole insect preparation, allows stable recordings within a working day. In comparison, EAGs on excised antennae have a lifetime of 2 hr. A custom hardware/software interface was developed between the EAG electrodes and a robot. The measurement system resolves individual odor patches up to 10 Hz, which exceeds the time scale of artificial chemical sensors12. The efficiency of EAG sensors for olfactory searches is further demonstrated in driving the robot toward a source of pheromone. By using identical olfactory stimuli and sensors as in real animals, our robotic platform provides a direct means for testing biological hypotheses about olfactory coding and search strategies13. It may also prove beneficial for detecting other odorants of interests by combining EAGs from different insect species in a bioelectronic nose configuration14 or using nanostructured gas sensors that mimic insect antennae15.

We describe a protocol for using insect antennae in the form of electroantennograms (EAGs) on autonomous robots. Our experimental design allows stable recordings within a day and resolves individual odor patches up to 10 Hz. The efficiency of EAG sensors for olfactory searches is demonstrated in driving a robot toward an odor source.

Robots designed to track chemical leaks in hazardous industrial facilities1 or explosive traces in landmine fields2 face the same problem as insects ...

Extraction and Quantification of Volatile Sex Pheromone Responses in C. elegans

Volatile Sex Pheromone Extraction and Chemoattraction Assay in Caenorhabditis elegans

Chemical communication is vital in organismal health, reproduction, and overall well-being. Understanding the molecular pathways, neural processes, and computations governing these signals remains an active area of research. The nematode Caenorhabditis elegans provides a powerful model for studying these processes as it produces a volatile sex pheromone. This pheromone is synthesized by virgin females or sperm-depleted hermaphrodites and serves as an attractant for males.
This protocol describes a detailed method for isolating the volatile sex pheromone from several C. elegans strains (WT strain N2, daf-22, and fog-2) and C. remanei. We also provide a protocol for quantifying the male chemotaxis response to the volatile sex pheromone. Our analysis utilizes measurements such as chemotaxis index (C.I.), arrival time (A.T.), and a trajectory plot to compare male responses under various conditions accurately. This method allows for robust comparisons between males of different genetic backgrounds or developmental stages. Furthermore, the experimental setup outlined here is adaptable to investigating other chemoattraction chemicals.

Author Spotlight: Examining Volatile Sex Pheromone Influence on Male C. elegans Behavior

This protocol establishes methods for extracting and quantifying responses to the volatile sex pheromone in C. elegans, providing tools to study chemical communication and navigation trajectory.

Chemical communication is vital in organismal health, reproduction, and overall well-being. Understanding the molecular pathways, neural processes, and ...