Name: a free-breathing fmri method to study human olfactory function
Uploaded: 2017-07-30T13:00:00
Description: Watch this Scientific Journal Video about A Free-breathing fMRI Method to Study Human Olfactory Function at JoVE.com

The authors have no acknowledgements.

The following experimental protocol followed the guidelines of the Institutional Review Board of the Pennsylvania State University College of Medicine, and the human subject gave written informed consent before participating in the study.
Note: For the purpose of demonstration, a simple odor stimulation paradigm using a commercially available, MRI-compatible olfactometer is presented. This paradigm has proven effective in reducing the habituation effect and has produced reliable olfactory fMRI...

<ol>
	<li>Doty, R. L., Reyes, P. F., Gregor, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presence+of+both+odor+identification+and+detection+deficits+in+Alzheimer's+disease.">Presence of both odor identification and detection deficits in Alzheimer's disease.</a> Brain Res Bull. 18 (5), 597-600 (1987).</li><li>Hummel, T., 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=Olfactory+FMRI+in+patients+with+Parkinson's+disease.">Olfactory FMRI in patients with Parkinson's disease.</a> Front Integr Neurosci. 4, 125 (2010).</li><li>Mesholam, R. I., Moberg, P. J., Mahr, R. N., Doty, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfaction+in+neurodegenerative+disease:+a+meta-analysis+of+olfactory+functioning+in+Alzheimer's+and+Parkinson's+diseases.">Olfaction in neurodegenerative disease: a meta-analysis of olfactory functioning in Alzheimer's and Parkinson's diseases.</a> Arch Neurol. 55 (1), 84-90 (1998).</li><li>Pause, B. M., Miranda, A., G&#246;der, R., Aldenhoff, J. B., Ferstl, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+olfactory+performance+in+patients+with+major+depression.">Reduced olfactory performance in patients with major depression.</a> J Psychiatr Res. 35 (5), 271-277 (2001).</li><li>Vasterling, J. J., Brailey, K., Sutker, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+identification+in+combat-related+posttraumatic+stress+disorder.">Olfactory identification in combat-related posttraumatic stress disorder.</a> J Trauma Stress. 13 (2), 241-253 (2000).</li><li>Anderson, A. K., 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=Dissociated+neural+representations+of+intensity+and+valence+in+human+olfaction.">Dissociated neural representations of intensity and valence in human olfaction.</a> Nat Neurosci. 6 (2), 196-202 (2003).</li><li>Gottfried, J. A., Deichmann, R., Winston, J. S., Dolan, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+heterogeneity+in+human+olfactory+cortex:+an+event-related+functional+magnetic+resonance+imaging+study.">Functional heterogeneity in human olfactory cortex: an event-related functional magnetic resonance imaging study.</a> J Neurosci. 22 (24), 10819-10828 (2002).</li><li>Sobel, N., 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=Sniffing+and+smelling:+separate+subsystems+in+the+human+olfactory+cortex.">Sniffing and smelling: separate subsystems in the human olfactory cortex.</a> Nature. 392 (6673), 282-286 (1998).</li><li>Sun, X., Wang, J., Weitekamp, C. W., Yang, Q. X. <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+Data+Processing+Method+for+Olfactory+fMRI+Examinations.">A Novel Data Processing Method for Olfactory fMRI Examinations.</a> Proc Intl Soc Mag Res Med. 18 (2010), 1161 (2010).</li><li>Zatorre, R. J., Jones-Gotman, M., Evans, A. C., Meyer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+localization+and+lateralization+of+human+olfactory+cortex.">Functional localization and lateralization of human olfactory cortex.</a> Nature. 360 (6402), 339-340 (1992).</li><li>Boley, J. C., Pontier, J. P., Smith, S., Fulbright, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facial+changes+in+extraction+and+nonextraction+patients.">Facial changes in extraction and nonextraction patients.</a> Angle Orthod. 68 (6), 539-546 (1998).</li><li>Furman, J. M., Koizuka, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reorientation+of+poststimulus+nystagmus+in+tilted+humans.">Reorientation of poststimulus nystagmus in tilted humans.</a> J Vestib Res. 4 (6), 421-428 (1994).</li><li>Loevner, L. A., Yousem, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overlooked+metastatic+lesions+of+the+occipital+condyle:+a+missed+case+treasure+trove.">Overlooked metastatic lesions of the occipital condyle: a missed case treasure trove.</a> Radiographics. 17 (5), 1111-1121 (1997).</li><li>Tabert, M. H., 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=Validation+and+optimization+of+statistical+approaches+for+modeling+odorant-induced+fMRI+signal+changes+in+olfactory-related+brain+areas.">Validation and optimization of statistical approaches for modeling odorant-induced fMRI signal changes in olfactory-related brain areas.</a> Neuroimage. 34 (4), 1375-1390 (2007).</li><li>Wang, J., Sun, X., Yang, Q. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+olfactory+fMRI+studies:+Implication+of+respiration.">Methods for olfactory fMRI studies: Implication of respiration.</a> Hum Brain Mapp. 35 (8), 3616-3624 (2014).</li><li>Gottfried, J. A., O'Doherty, J., Dolan, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appetitive+and+aversive+olfactory+learning+in+humans+studied+using+event-related+functional+magnetic+resonance+imaging.">Appetitive and aversive olfactory learning in humans studied using event-related functional magnetic resonance imaging.</a> J Neurosci. 22 (24), 10829-10837 (2002).</li><li>Popp, R., Sommer, M., M&#252;ller, J., Hajak, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactometry+in+fMRI+studies:+odor+presentation+using+nasal+continuous+positive+airway+pressure.">Olfactometry in fMRI studies: odor presentation using nasal continuous positive airway pressure.</a> Acta Neurobiol Exp (Wars). 64 (2), 171-176 (2004).</li><li>Wang, J., 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=Olfactory+Habituation+in+the+Human+Brain.">Olfactory Habituation in the Human Brain.</a> Proc Intl Soc Mag Res Med. 20, 2150 (2012).</li><li>Grunfeld, 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=The+responsiveness+of+fMRI+signal+to+odor+concentration).">The responsiveness of fMRI signal to odor concentration).</a> , A237-A238 (2005).</li><li>Jia, H., 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=Functional+MRI+of+the+olfactory+system+in+conscious+dogs.">Functional MRI of the olfactory system in conscious dogs.</a> PLoS One. 9 (1), e86362 (2014).</li><li>Karunanayaka, P., 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=Networks+involved+in+olfaction+and+their+dynamics+using+independent+component+analysis+and+unified+structural+equation+modeling.">Networks involved in olfaction and their dynamics using independent component analysis and unified structural equation modeling.</a> Hum Brain Mapp. 35 (5), 2055-2072 (2014).</li><li>Royet, J. P., 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=Functional+neuroanatomy+of+different+olfactory+judgments.">Functional neuroanatomy of different olfactory judgments.</a> Neuroimage. 13 (3), 506-519 (2001).</li><li>Doty, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+age+and+age-related+diseases+on+olfactory+function.">Influence of age and age-related diseases on olfactory function.</a> Ann N Y Acad Sci. 561, 76-86 (1989).</li><li>Wang, J., 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=Olfactory+deficit+detected+by+fMRI+in+early+Alzheimer's+disease.">Olfactory deficit detected by fMRI in early Alzheimer's disease.</a> Brain Res. 1357, 184-194 (2010).</li><li>Moessnang, C., 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=Altered+activation+patterns+within+the+olfactory+network+in+Parkinson's+disease.">Altered activation patterns within the olfactory network in Parkinson's disease.</a> Cereb Cortex. 21 (6), 1246-1253 (2011).</li><li>Vasavada, M. 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=Olfactory+cortex+degeneration+in+Alzheimer's+disease+and+mild+cognitive+impairment.">Olfactory cortex degeneration in Alzheimer's disease and mild cognitive impairment.</a> J Alzheimers Dis. 45 (3), 947-958 (2015).</li><li>Jacobs, H. I., Radua, J., L&#252;ckmann, H. C., Sack, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meta-analysis+of+functional+network+alterations+in+Alzheimer's+disease:+toward+a+network+biomarker.">Meta-analysis of functional network alterations in Alzheimer's disease: toward a network biomarker.</a> Neurosci Biobehav Rev. 37 (5), 753-765 (2013).</li><li>Murphy, C., Cerf-Ducastel, B., Calhoun-Haney, R., Gilbert, P. E., Ferdon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ERP,+fMRI+and+functional+connectivity+studies+of+brain+response+to+odor+in+normal+aging+and+Alzheimer's+disease.">ERP, fMRI and functional connectivity studies of brain response to odor in normal aging and Alzheimer's disease.</a> Chem Senses. 30 Suppl 1, i170-i171 (2005).</li><li>Hummel, T., Kobal, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+human+evoked+potentials+related+to+olfactory+or+trigeminal+chemosensory+activation.">Differences in human evoked potentials related to olfactory or trigeminal chemosensory activation.</a> Electroen Clin Neuro. 84 (1), 84-89 (1992).</li><li>Cerf-Ducastel, B., Murphy, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FMRI+brain+activation+in+response+to+odors+is+reduced+in+primary+olfactory+areas+of+elderly+subjects.">FMRI brain activation in response to odors is reduced in primary olfactory areas of elderly subjects.</a> Brain Res. 986 (1-2), 39-53 (2003).</li><li>Cain, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+the+trigeminal+nerve+to+perceived+odor+magnitude.">Contribution of the trigeminal nerve to perceived odor magnitude.</a> Ann NY Acad Sci. 237, 28-34 (1974).</li><li>Murphy, C., Gilmore, M. M., Seery, C. S., Salmon, D. P., Lasker, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+thresholds+are+associated+with+degree+of+dementia+in+Alzheimer's+disease.">Olfactory thresholds are associated with degree of dementia in Alzheimer's disease.</a> Neurobiol Aging. 11 (4), 465-469 (1990).</li><li>Doty, R. L., Brugger, W. E., Jurs, P. C., Orndoff, M. A., Snyder, P. J., Lowry, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intranasal+trigeminal+stimulation+from+odorous+volatiles:+Psychometric+responses+from+anosmic+and+normal+humans.">Intranasal trigeminal stimulation from odorous volatiles: Psychometric responses from anosmic and normal humans.</a> Physiol Behav. 20 (2), 175-185 (1978).</li><li>Kobal, G., Hummel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+and+intranasal+trigeminal+event-related+potentials+in+anosmic+patients.">Olfactory and intranasal trigeminal event-related potentials in anosmic patients.</a> Laryngoscope. 108 (7), 1033-1035 (1998).</li><li>Frasnelli, J., Lundstr&#246;m, J. N., Sch&#246;pf, V., Negoias, S., Hummel, T., Lepore, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+processing+streams+in+chemosensory+perception.">Dual processing streams in chemosensory perception.</a> Front Hum Neurosci. 6, (2012).</li><li>Yousem, D. 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=Gender+effects+on+odor-stimulated+functional+magnetic+resonance+imaging.">Gender effects on odor-stimulated functional magnetic resonance imaging.</a> Brain Res. 818 (2), 480-487 (1999).</li><li>Koulivand, P. H., Ghadiri, M. K., Gorji, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lavender+and+the+nervous+system.">Lavender and the nervous system.</a> Evid Based Compl Alt Med. 2013, (2013).</li><li>Yousem, D. 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=Functional+MR+imaging+during+odor+stimulation:+Preliminary+data.">Functional MR imaging during odor stimulation: Preliminary data.</a> Neuroradiology. 204 (3), 833-838 (1997).</li><li>Hummel, T., Doty, R. L., Yousem, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+MRI+of+intranasal+chemosensory+trigeminal+activation.">Functional MRI of intranasal chemosensory trigeminal activation.</a> Chem Senses. 30 (suppl. 1), i205-i206 (2005).</li></ol>

Experimental procedures should be considered carefully and executed properly for collection of reliable olfactory activation data. The critical steps within the protocol include implementing a respiration-triggered paradigm to synchronize odor delivery with image acquisition, preparing proper concentrations of odorants to control psychophysical responses, setting up the olfactometer with reliable stable respiration signal and constant air flow, and post-processing respiration and odor administration timing data using ONS...

The human olfactory system is understood to be much more than a sensory system because olfaction also plays an important role in homeostatic regulation and emotions. Clinically, the human olfactory system is known to be vulnerable to attacks of many prevalent neurological diseases and psychiatric disorders, such as Alzheimer&#39;s disease, Parkinson&#39;s disease, post-traumatic stress disorder, and depression1,2,3,4,5. Currently, functional magnetic resonance imaging (fMRI) with blood-oxygen-level-dependent (BOLD) contrast is the most valuable technique for mapping functions of the human brain. A significant amount of knowledge about specific functions of central olfactory structures (e.g., piriform cortex, orbitofrontal cortex, amygdala, and insular cortex) has been acquired with this technique6,7,8,9,10.
The application of fMRI to studies of the human central olfactory system and associated diseases, however, has been hindered by two major obstacles: rapid habituation of BOLD signal and variable modulation by respiration. In everyday life, when exposed to an odorant for a period of time, we quickly habituate to the scent. In fact, when studied using olfactory fMRI, the odor-induced fMRI signal is rapidly attenuated by habituation, which poses a challenge on stimulation paradigm designs8,10,11,12,13,14. The initial&#160;significant BOLD signal in the primary olfactory cortex only persists for several seconds after odorant onset. Therefore, olfactory fMRI paradigms should avoid prolonged or frequent odor stimulations in a short period of time. To reduce the habituation effect, some studies have attempted to present alternating odors in an fMRI paradigm. However, this approach may complicate data analysis since each odorant can be treated as an independent stimulation event.
Another technical issue arises with variability in subjects&#39; respiration patterns; inhalation does not always synchronize with odorant administration during a fixed-timing paradigm. The onset and duration of olfactory stimulation are modulated by each individual&#39;s respiration, which confounds fMRI data quality and analysis. Some studies have attempted to mitigate this problem with visual or auditory cues to synchronize breathing and odorant onset, but the compliance of subjects is variable, especially in the clinical population. The brain activations associated with these cues could also complicate data analysis in certain applications. Thus, synchronizing inhalation with odorant delivery can be crucial for olfactory fMRI studies15.
An additional consideration vital to olfactory fMRI, especially in the data analysis process, is odorant selection. Finding an appropriate odorant concentration with respect to perceived intensity is important for quantification and comparison of activation levels in the brain under various experimental conditions or diseases. Odorant selection must also take into consideration odor valence, or pleasantness. This is known to cause divergent temporal profiles in olfactory learning16,17. Lavender odor was chosen for this demonstration partially for this reason. Depending on the purpose of a specific study, different odorants may be better choices. In addition, trigeminal stimulation must be minimized to reduce activation not directly related to olfaction18.
In this report, we demonstrate an fMRI technique to set up and run a respiration-triggered paradigm using an olfactometer in the magnetic resonance environment. We also present a post-processing tool that can diminish some timing errors that may have occurred during data acquisition in an attempt to further improve data analysis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3T MR scanner</td>
			<td>Siemens</td>
			<td></td>
			<td>Any MR scanner is acceptable.&#160;</td>
		</tr>
		<tr>
			<td>Olfactometer</td>
			<td>Emerging Tech Trans, LLC</td>
			<td></td>
			<td>Any olfactometer with similar capabilities is acceptable.</td>
		</tr>
		<tr>
			<td>6-channel odorant carrier</td>
			<td>Emerging Tech Trans, LLC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nosepiece/applicator</td>
			<td>Emerging Tech Trans, LLC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PTFE tubing</td>
			<td>Emerging Tech Trans, LLC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TTL convertor box</td>
			<td>Emerging Tech Trans, LLC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Respiratory sensor belt</td>
			<td>Emerging Tech Trans, LLC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lavender oil</td>
			<td>Givaudan Flavors Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1,2 propanediol</td>
			<td>Sigma</td>
			<td>P6209</td>
			<td></td>
		</tr>
		<tr>
			<td>ONSET</td>
			<td></td>
			<td></td>
			<td>www.pennstatehershey.org/web/nmrlab/resources/software/onset</td>
		</tr>
		<tr>
			<td>SPM8&#160;</td>
			<td>Wellcome Trust Center for Neuroimaging, University College London, London, UK&#160;</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a free-breathing fmri method to study human olfactory function

The study of human olfaction is a highly complex and valuable field with applications ranging from biomedical research to clinical evaluation. Currently, evaluation of the functions of the human central olfactory system with functional magnetic resonance imaging (fMRI) is still a challenge because of several technical difficulties. There are some significant variables to take into account when considering an effective method for mapping the function of the central olfactory system using fMRI, including proper odorant selection, the interaction between odor presentation and respiration, and potential anticipation of or habituation to odorants. An event-related, respiration-triggered olfactory fMRI technique can accurately administer odorants to stimulate the olfactory system while minimizing potential interference. It can effectively capture the precise onsets of fMRI signals in the primary olfactory cortex using our data post-processing method. The technique presented here provides an efficient and practical means for generating reliable olfactory fMRI results. Such a technique can ultimately be applied in the clinical realm as a diagnostic tool for diseases associated with olfactory degeneration, including Alzheimer&#39;s and Parkinson&#39;s disease, as we begin to further understand the complexities of the human olfactory system.

Figure 1 demonstrates the set-up of olfactory fMRI inside and outside of the magnet room, taking into account MR-compatibility. Figure 2a demonstrates a standard fixed-timing paradigm, while Figure 2b demonstrates a paradigm where the &#34;respiration trigger&#34; allows for the synchronization of odor delivery and inhalation.
A regular res...

Watch this Scientific Journal Video about A Free-breathing fMRI Method to Study Human Olfactory Function at JoVE.com

A Free-breathing fMRI Method to Study Human Olfactory Function

We present the technical challenges and solutions for obtaining reliable functional magnetic resonance imaging (fMRI) data from the human central olfactory system. This includes special considerations in olfactory fMRI paradigm design, descriptions of fMRI data acquisition with an MRI-compatible olfactometer, odorant selection, and a special software tool for data post-processing.

The study of human olfaction is a highly complex and valuable field with applications ranging from biomedical research to clinical evaluation. Currently, ...

The overall goal of this video is to present and explain an efficient and easily adaptable method of fMRI in order to effectively explore the mapping of the human olfactory system without interference of natural breathing. One obstacle of using olfactory fMRI to study the brain activation responding to odor and stimulation, is the inconsistency of repetitive odor stimulation, which is caused by the asynchrony of respiration and odor delivery. As illustrated here, when the odor presentation in a fixed timing odor stimulation paradigm is not synchronized with respiration, the repetitive odor stimulation can happen at different phases of respiration.A solution to this problem is to set up a respiration-triggered olfactory fMRI paradigm. Effective olfactory fMRI data collection is accomplished by first designing an event related respiration-triggered olfactory stimulation paradigm on the olfactometer that is resist to habituation and inconsistent inhalation patterns. The second step is to prepare odorants for the olfactory stimulation, taking care to use odorants that minimize trigeminal stimulation and habituation effects.The third step is to set up and run the experiment in the MRI environment by assembling the olfactometer with the capability of running a respiration-triggered paradigm. The final step is to process the data using the program ONSET to define the actual onset and the duration vectors of the stimuli. Ultimately, this technique has the potential to facilitate more sensitive exploration and connections of the human olfactory system.Here, we will demonstrate how to set up a respiration-triggered olfactory fMRI experiment using an ETT Olfactometer. To create a paradigm, assign the odorant channels to specific odorants. There are six odorant channels available in this olfactometer system.Input and edit the sequence and timing of open and close of these channels. Push the Paradigm Setting button on the touchscreen. To input or edit the sequence of the open and close of the odorant channels, push the Edit button for the Valve Sequence.When finished, push the Edit button again to exit the editing mode. To input or edit the timing parameters for the open and close of the odorant channels, push the Set button for Time Sequence. When finished, push the Set button again to exit the editing mode.After finishing the Paradigm Setting, push the Return button to return to the main window. Demonstrated in this video is a simple stimulation paradigm of the same odorant in the same concentration. Each odorant presentation is interleaved with the presentation of fresh air.In this paradigm, the same odor is presented 12 times. Each time, the odorant presentation lasts for six seconds with variable amounts of time between the odor presentations. Set the Valve Open and Valve Close durations, accordingly.In this demonstration, one concentration of lavender oil is prepared. We use lavender oil because of its minimal trigeminal stimulation and activation of certain brain areas. The odorant concentrations can be varied depending on the odorant selected.Choose the concentrations based upon the ideal perception threshold of the odor. By varying the concentration, we lessen the likelihood of habituation to an odorant. To prepare the concentrations, mix the lavender oil with propylene glycol.Propylene glycol is a commonly used solvent for odorants and is stable and nearly odorless. After mixing all solutions as outlined in the paradigm design, pour the same amount of odorants into each container bottle. Then, attach the bottles to the odorant carrier in the correct order.To set up the olfactometer in the MRI, place the odorant carrier in the magnet room next to the machine. Run the tubes through the wave guide to connect to the carrier, taking care that the order of tubes matches the order connecting to the olfactometer. For ease, we color code our tubings to match the odorant channels.Turn the machine on. Adjust the main airflow, as necessary, as well as the individual flow rates for the carrier and flush. Then, connect the respiration belt to the olfactometer and run the belt through the wave guide.The belt will record the subject's respiration activity, as well as the utilization of the respiration trigger. Bring the subject into the magnet room and place the face mask on the subject. When the subject is lying down, place the respiratory belt around their chest or abdomen where there is clear respiration event.Check the respiration pattern on the screen of the olfactometer. If the waves plateau, the belt is fastened too tightly and the respiration trigger will not work properly. Adjust the belt until a complete respiration trace can be seen, as clear respiration peak signals are critical for a successful execution of a respiration-triggered paradigm.Create a data folder on the olfactometer to store the respiration data. The synchrony of the odor presentation with respiration should be tested for accuracy by using the Paradigm Check and adjusting the Trigger Delay time, as necessary. Push the Paradigm Check button on the touchscreen and monitor if odor delivery happens during the inhalation phase of the respiration cycle.The odor delivery period is highlighted in the color pink on the respiration trace. If odor delivery starts during the exhalation phase, to back to Paradigm Setting, adjust the Trigger Delay in the time sequence and then redo the Paradigm Check. Set the paradigm to Trigger In mode so that the execution of the odor stimulation paradigm will be triggered by MRI scanning.Then select the Respiration Trigger Start function. The paradigm is now ready to be run. Once the odor stimulation paradigm is completed, check the respiration data saved in the olfactometer.This file includes the respiration trace and the time stamp of each functional MRI image, respiration trigger, open and close of each odor channel, and the subject response, using a response device. It can be processed using the program ONSET to acquire the actual stimulation onset vectors and durations for fMRI data processing. ONSET can be run in the IDL Virtual Machine.To get the odor stimulation onset vectors and durations, click Respiration Validation. Load the respiration data file that's saved from the olfactometer and set the post processing parameters for ONSET processing. Run ONSET.The actual stimulation onset vectors, the start of each inhalation during odor delivery, will automatically be detected based on the timing of odor presentation and the respiration trace. The results, including the onset and duration vectors, can be utilized in fMRI data processing. Process the fMRI data following standard procedures.Here is an example of data processing using SPM8. Use either the image numbers or the onset vectors and durations in seconds from the onset output, depending on the timing unit chosen for the SPM paradigm design. Generate a statistical parametric map at the individual level by fitting the stimulation paradigm using the actual onset and duration vectors to the functional data with the canonical hemodynamic response function.Here is an example of the primary olfactory cortex activation responding to the odor stimulation. In contrast to the respiration-triggered odor stimulation paradigm, for example, a fixed timing paradigm which we can see here from the same subject, the odor delivery is not triggered by respiration activity. Since there is no control of the subject's respiration, there is significant variability in the amount of odorant that is inhaled.Following the standard procedure to process these fMRI data with the same statistical threshold as that used for the previous respiration-triggered fMRI data, no significant activation was detected in the primary olfactory cortex. However, when a respiration trigger is implemented, odorant presentation is always synchronized with inhalation, improving the consistency and accuracy of odor stimulation. After watching this video, you should have a good understanding of the current practice of olfactory fMRI using an event related respiration-triggered paradigm.Once the technique demonstrated in this video has been mastered, further olfactory stimulation paradigm design and analysis methods can be applied to map the functions of the human central olfactory system and to further explore the olfactory deficits in neurodegenerative diseases.

a-free-breathing-fmri-method-to-study-human-olfactory-function

Research

JoVE Journal

Neuroscience

Combining Transcranial Magnetic Stimulation and fMRI to Examine the Default Mode Network

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful rest."1,2,3 It is thought the default mode network corresponds to self-referential or "internal mentation".2,3
It has been hypothesized that, in humans, activity within the default mode network is correlated with certain pathologies (for instance, hyper-activation has been linked to schizophrenia 4,5,6 and autism spectrum disorders 7 whilst hypo-activation of the network has been linked to Alzheimer's and other neurodegenerative diseases 8). As such, noninvasive modulation of this network may represent a potential therapeutic intervention for a number of neurological and psychiatric pathologies linked to abnormal network activation. One possible tool to effect this modulation is Transcranial Magnetic Stimulation: a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.9
In order to explore the default mode network's propensity towards and tolerance of modulation, we will be combining TMS (to the left inferior parietal lobe) with functional magnetic resonance imaging (fMRI). Through this article, we will examine the protocol and considerations necessary to successfully combine these two neuroscientific tools.

In this article, we examine the methodology and considerations relevant to the combination of TMS and fMRI to examine the effects of brain stimulation on the default network.

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful ...

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the olfactory bulb (OB). The molecular mechanisms of OSN axon growth and targeting are not well understood. Manipulating gene expression and subsequent 
visualizing of single OSN axons and their terminal arbor morphology have thus far been challenging. To study gene function at the single cell level within a specified 
time frame, we developed a lentiviral based technique to manipulate gene expression in OSNs in vivo. Lentiviral particles are delivered to OSNs by microinjection 
into the olfactory epithelium (OE). Expression cassettes are then permanently integrated into the genome of transduced OSNs. Green fluorescent protein expression 
identifies infected OSNs and outlines their entire morphology, including the axon terminal arbor. Due to the short turnaround time between microinjection and 
reporter detection, gene function studies can be focused within a very narrow period of development. With this method, we have detected GFP expression within as 
few as three days and as long as three months following injection. We have achieved both over-expression and shRNA mediated knock-down by lentiviral microinjection. 
This method provides detailed morphologies of OSN cell bodies and axons at the single cell level in vivo, and thus allows characterization of candidate gene function 
during olfactory development.

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

Correlating Behavioral Responses to fMRI Signals from Human Prefrontal Cortex: Examining Cognitive Processes Using Task Analysis

The aim of this methods paper is to describe how to implement a neuroimaging technique to examine complementary brain processes engaged by two similar tasks. Participants' behavior during task performance in an fMRI scanner can then be correlated to the brain activity using the blood-oxygen-level-dependent signal. We measure behavior to be able to sort correct trials, where the subject performed the task correctly and then be able to examine the brain signals related to correct performance. Conversely, if subjects do not perform the task correctly, and these trials are included in the same analysis with the correct trials we would introduce trials that were not only for correct performance. Thus, in many cases these errors can be used themselves to then correlate brain activity to them. We describe two complementary tasks that are used in our lab to examine the brain during suppression of an automatic responses: the stroop1 and anti-saccade tasks. The emotional stroop paradigm instructs participants to either report the superimposed emotional 'word' across the affective faces or the facial 'expressions' of the face stimuli1,2. When the word and the facial expression refer to different emotions, a conflict between what must be said and what is automatically read occurs. The participant has to resolve the conflict between two simultaneously competing processes of word reading and facial expression. Our urge to read out a word leads to strong 'stimulus-response (SR)' associations; hence inhibiting these strong SR's is difficult and participants are prone to making errors. Overcoming this conflict and directing attention away from the face or the word requires the subject to inhibit bottom up processes which typically directs attention to the more salient stimulus. Similarly, in the anti-saccade task3,4,5,6, where an instruction cue is used to direct only attention to a peripheral stimulus location but then the eye movement is made to the mirror opposite position. Yet again we measure behavior by recording the eye movements of participants which allows for the sorting of the behavioral responses into correct and error trials7 which then can be correlated to brain activity. Neuroimaging now allows researchers to measure different behaviors of correct and error trials that are indicative of different cognitive processes and pinpoint the different neural networks involved.

The goal of our research is to correlate behavior to brain activity. Accurate behavioral measures and imaging techniques allow us to elucidate brain-behavior relationships.

The aim of this methods paper is to describe how to implement a neuroimaging technique to examine complementary brain processes engaged by two similar ...

Co-analysis of Brain Structure and Function using fMRI and Diffusion-weighted Imaging

The study of complex computational systems is facilitated by network maps, such as circuit diagrams. Such mapping is particularly informative when studying the brain, as the functional role that a brain area fulfills may be largely defined by its connections to other brain areas. In this report, we describe a novel, non-invasive approach for relating brain structure and function using magnetic resonance imaging (MRI). This approach, a combination of structural imaging of long-range fiber connections and functional imaging data, is illustrated in two distinct cognitive domains, visual attention and face perception. Structural imaging is performed with diffusion-weighted imaging (DWI) and fiber tractography, which track the diffusion of water molecules along white-matter fiber tracts in the brain (Figure 1). By visualizing these fiber tracts, we are able to investigate the long-range connective architecture of the brain. The results compare favorably with one of the most widely-used techniques in DWI, diffusion tensor imaging (DTI). DTI is unable to resolve complex configurations of fiber tracts, limiting its utility for constructing detailed, anatomically-informed models of brain function. In contrast, our analyses reproduce known neuroanatomy with precision and accuracy. This advantage is partly due to data acquisition procedures: while many DTI protocols measure diffusion in a small number of directions (e.g., 6 or 12), we employ a diffusion spectrum imaging (DSI)1, 2 protocol which assesses diffusion in 257 directions and at a range of magnetic gradient strengths. Moreover, DSI data allow us to use more sophisticated methods for reconstructing acquired data. In two experiments (visual attention and face perception), tractography reveals that co-active areas of the human brain are anatomically connected, supporting extant hypotheses that they form functional networks. DWI allows us to create a "circuit diagram" and reproduce it on an individual-subject basis, for the purpose of monitoring task-relevant brain activity in networks of interest.

We describe a novel approach for simultaneous analysis of brain function and structure using magnetic resonance imaging (MRI). We assess brain structure with high-resolution diffusion-weighted imaging and white-matter fiber tractography. Unlike standard structural MRI, these techniques allow us to directly relate anatomical connectivity to functional properties of brain networks.

The study of complex computational systems is facilitated by network maps, such as circuit diagrams. Such mapping is particularly informative when ...

Appetitive Associative Olfactory Learning in Drosophila Larvae

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

Appetitive Associative Olfactory Learning in Drosophila Larvae

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

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

Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disorders

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium carbonate. Animal studies as well as human genetic studies indicate that lithium affects molecular targets that are involved in neuronal growth, survival and maturation, and notably molecules involved in Wnt signaling. Given the ethical challenge to obtaining brain biopsies for investigating dynamic molecular changes associated with lithium-response in the central nervous system (CNS), one may consider the use of neurons obtained from olfactory tissues to achieve this goal.The olfactory epithelium contains olfactory receptor neurons at different stages of development and glial-like supporting cells. This provides a unique opportunity to study dynamic changes in the CNS of patients with neuropsychiatric diseases, using olfactory tissue safely obtained from nasal biopsies. To overcome the drawback posed by substantial contamination of biopsied olfactory tissue with non-neuronal cells, a novel approach to obtain enriched neuronal cell populations was developed by combining nasal biopsies with laser-capture microdissection. In this study, a system for investigating treatment-associated dynamic molecular changes in neuronal tissue was developed and validated, using a small pilot sample of BD patients recruited for the study of the molecular mechanisms of lithium treatment response.

In this study, a novel platform to investigate intraneuronal molecular signatures of treatment response in bipolar disorder (BD) was developed and validated. Olfactory epithelium from BD patients was obtained through nasal biopsies. Then laser-capture microdissection was combined with Real Time RT PCR to investigate the molecular signature of lithium response in BD.

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

A Protocol for the Administration of Real-Time fMRI Neurofeedback Training

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control neuroplastic processes and correct abnormal function, or even shift functions from damaged tissue to physiologically healthy brain regions, hold the potential to dramatically improve overall health. Of the current neuroplastic interventions in development, neurofeedback training (NFT) from functional Magnetic Resonance Imaging (fMRI) has the advantages of being completely non-invasive, non-pharmacologic, and spatially localized to target brain regions, as well as having no known side effects. Furthermore, NFT techniques, initially developed using fMRI, can often be translated to exercises that can be performed outside of the scanner without the aid of medical professionals or sophisticated medical equipment. In fMRI NFT, the fMRI signal is measured from specific regions of the brain, processed, and presented to the participant in real-time. Through training, self-directed mental processing techniques, that regulate this signal and its underlying neurophysiologic correlates, are developed. FMRI NFT has been used to train volitional control over a wide range of brain regions with implications for several different cognitive, behavioral, and motor systems. Additionally, fMRI NFT has shown promise in a broad range of applications such as the treatment of neurologic disorders and the augmentation of baseline human performance. In this article, we present an fMRI NFT protocol developed at our institution for modulation of both healthy and abnormal brain function, as well as examples of using the method to target both cognitive and auditory regions of the brain.

The ability to induce and/or control neural plasticity may be critical in future treatments for neurologic disorders and the recovery from brain injury. In this paper, we present a protocol on the use of neurofeedback training with functional magnetic resonance imaging to modulate human brain function.

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control ...

New Methods to Study Gustatory Coding

The sense of taste allows animals to detect chemicals in the environment, giving rise to behaviors critical for survival. When Gustatory Receptor Neurons (GRNs) detect tastant molecules, they encode information about the identity and concentration of the tastant as patterns of electrical activity that then propagate to follower neurons in the brain. These patterns constitute internal representations of the tastant, which then allow the animal to select actions and form memories. The use of relatively simple animal models has been a powerful tool to study basic principles in sensory coding. Here, we propose three new methods to study gustatory coding using the moth Manduca sexta. First, we present a dissection procedure for exposing the maxillary nerves and the subesophageal zone (SEZ), allowing recording of the activity of GRNs from their axons. Second, we describe the use of extracellular electrodes to record the activity of multiple GRNs by placing tetrode wires directly into the maxillary nerve. Third, we present a new system for delivering and monitoring, with high temporal precision, pulses of different tastants. These methods allow the characterization of neuronal responses in vivo directly from GRNs before, during and after tastants are delivered. We provide examples of voltage traces recorded from multiple GRNs, and present an example of how a spike sorting technique can be applied to the data to identify the responses of individual neurons. Finally, to validate our recording approach, we compare extracellular recordings obtained from GRNs with tetrodes to intracellular recordings obtained with sharp glass electrodes.

We present three new methods to study gustatory coding. Using a simple animal, the moth Manduca sexta (Manduca), we describe a dissection protocol, the use of extracellular tetrodes to record the activity of multiple gustatory receptor neurons, and a system for delivering and monitoring precisely timed pulses of tastants.

The sense of taste allows animals to detect chemicals in the environment, giving rise to behaviors critical for survival. When Gustatory Receptor Neurons ...

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a considerable amount of information has been generated regarding ORN responses to odorants, the role of specific ORNs in driving behavioral responses remains poorly understood. Complications in behavior analyses arise due to different volatilities of odorants that activate individual ORNs, multiple ORNs activated by single odorants, and the difficulty in replicating naturally observed temporal variations in olfactory stimuli using conventional odor-delivery methods in the laboratory. Here, we describe a protocol that analyzes Drosophila larval behavior in response to simultaneous optogenetic stimulation of its ORNs. The optogenetic technology used here allows for specificity of ORN activation and precise control of temporal patterns of ORN activation. Corresponding larval movement is tracked, digitally recorded, and analyzed using custom written software. By replacing odor stimuli with light stimuli, this method allows for a more precise control of individual ORN activation in order to study its impact on larval behavior. Our method could be further extended to study the impact of second-order projection neurons (PNs) as well as local neurons (LNs) on larval behavior. This method will thus enable a comprehensive dissection of olfactory circuit function and complement studies on how olfactory neuron activities translate in to behavior responses.

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

This protocol analyzes navigational behavior of Drosophila larva in response to simultaneous optogenetic stimulation of its olfactory neurons. Light of 630 nm wavelength is used to activate individual olfactory neurons expressing a red-shifted channel rhodopsin. Larval movement is simultaneously tracked, digitally recorded, and analyzed using custom-written software.