This work was supported by the "Agence Nationale de la Recherche" grant ANR-09-JCJC-0117-01 and "Neurop&ocirc;le de Recherche Francilien-NERF" grant for Romain Chery. We thank Fran&ccedil;oise Lefebvre for the software development in C++/Qt, and Laurent Pinot and Batiste Janvier for help in the development of the optical imaging setup.

1. Preparing the animal for imaging (in accordance with European recommendations for care and use of laboratory animals, 86/609/EEC directive)
<ol>
	<li>6 to 8 weeks old C57BL6 male mice are anesthetized with a cocktail of ketamine (10mg/kg) and xylazine (100mg/kg) injected intraperitonealy. Surgery begins when the mouse no longer responds to hindpaw pinch. During the entire experiment the animal is placed on a heating pad. Body temperature is continuously monitored and maintained at 37&deg;C. Depth of anesthesia is maintained throughout surgery and imaging session by checking out the absence of limb withdraw. A subcutaneous injection of 20% of the initial anesthetic cocktail is otherwise administered.</li>
	<li>Using clippers, remove the hair from the scalp. Clean the exposed skin from residual hair by using sterile gauze soaked with saline.</li>
	<li>Place the mouse in the stereotaxic frame. The snout has to be in the same plane as the back of the head, in order to set the surface of the olfactory bulb to the horizontal. Secure firmly the ear and nose bars in order to prevent movements during imaging.</li>
	<li>Apply ophthalmic ointment on the animal's eyes to prevent drying and pain.</li>
	<li>Disinfect all surgical instruments with 70&deg; ethanol and the scalp area with successive sweeps of betadine.</li>
	<li>To remove the skin covering the skull, start by making an incision in the skin with scissors at the back of the head between the ears. Then cut in both sides towards the base of the ear and in the anteroposterior direction towards the forehead along the eyelids. Finish removing the scalp by cutting the skin on top of the snout close to the nose bar.</li>
	<li>Under binocular observation, use a cotton swab soaked with saline to gently detach the periosteum on the top of the skull. Use a pair of forceps to remove the remaining tissue and scrape the surface of the skull with a scalpel to have a clean preparation.</li>
	<li>The OB is a symmetric structure composed of two hemibulbs which are located between the eyelids. They are limited in the rostral and in the caudal direction by a venous sinus, and separated by the sagittal suture. Place a piece of absorbable gelatin sponge soaked with distilled water on the bone above the OB. It is important to keep this bone area moist throughout the experiment.</li>
</ol>
2. Preparing the cranial window
<ol>
	<li>Remove the gelatin sponge and start by gently scraping the bone with n&deg;10 scalpel blade. Keep a constant angle of 45&deg; between the blade and the bone, and move the blade from the eyelids to the sagittal side of the bulb area. Don't apply vertical pressure on the bone or scratch the bone above the venous sinus.</li>
	<li>During the thinning process, stop every 5min and place a hydrated gelatin sponge on the bone to cool down the preparation. Swab the skull with the sponge to remove bone dust.</li>
	<li>Keep sweeping and cooling alternatively until visualizing the trabeculae, the spongy bone layer. The fine vasculature of the OB must be visible at this stage. Stop scratching the bone, and start using the scalpel's tip perpendicularly to "draw" a rectangular area enclosing the OB. At this stage n&deg;11 scalpel blade can be used. Keep the surgery within the limits of the venous sinuses that should be safe of any scalpel stroke.</li>
	<li>Dig gradually the formed rectangular trench by using successive motions of the scalpel. Wipe the tip of the scalpel periodically to clean it and keep it sharp. Be extra cautious of the depth of the tip to avoid touching the dura surface.</li>
	<li>In order to get a sense of the thickness of the remaining bone, push it gently with the tip of a pair of forceps. If the bone flap folds under pressure, move to the next step.</li>
	<li>Under a drop of saline, use the tip of the scalpel oriented horizontally to lift up the bone flap. The removal of the flap has to be done carefully to avoid tearing off the remaining bone.</li>
	<li>Once the OB's surface is exposed, check for the absence of any bleeding or blood vessels anastomosis. Injuring the dura or the tissue surface will reduce the chances of obtaining optical signals. Wipe the area with a gelatin sponge soaked in saline in order to keep the bulb moist.</li>
	<li>Apply polyacrylate dental cement to form a well on the bone around the window.</li>
	<li>Place a drop of low melting point agarose (1.2%) over the dura, and put a sterile cover glass at the dimensions of the window. During the imaging session, a small quantity of agarose can be added in order to compensate the desiccation. Agarose will prevent the OB from moving with respiration and will provide a flat surface for optical imaging.</li>
</ol>
3. Optical imaging setup for olfactory activity mapping
<ol>
	<li>The olfactory stimulation must be precisely defined in time and intensity by the use of an olfactometer. We use a custom modified version of multivial perfusion system Valvebank 8II from Automate Scientific associated with a basic air compressor (compressed breathable air would be suited as well). This system allows for accurate and fast external valve control. Solutions of pure odorant are diluted in mineral oil at the chosen concentration. To activate the dorsal OB aldehydes such as hexanal are commonly used. A precise volume of the diluted odorant (20 to 50&mu;l) is loaded on a filter paper and placed in a syringe reservoir. Through the perfusion system, pressure controlled air is delivered to the system, ensuring a constant rate of odorized air flux delivery to the animal nose during valve opening. Odorant is delivered through a Tygon R-3603 Vacuum Tubing (Saint-Gobain Corporation) carrying air at a flow rate of &tilde;1000ml/min. Avoid contamination between odorants and residual quantities of odorant in the tubing. If available, reproducibility of the odorant stimulus can be controlled through the use of a flame ionization detector (microFID 2020 Photovac).</li>
	<li>The optical setup is turned on. It consists of a cooled interlaced 12 bits CCD camera (ORCA AG Hamamatsu) associated with a fluorescence stereomicroscope (Leica MZ16), a computer controlled olfactometer and stabilized excitation lamps with appropriate bandpass interference filters. A scheme describing our setup is provided in figure 2. For Intrinsic Optical Signals Imaging (IOSI), a 200W tungsten halogen lamp (Oriel QTH) coupled with a fiber ring light (Schott) plugged around the microscope objective are used to provide stable and even illumination. For Flavoprotein Autofluorescence Signals Imaging (FASI), a 150W metal Halide lamp (Leica) with a 5mm core liquid light guide provide even localized excitation of the fluorescence through the epi-illumination port of the stereomicroscope.
	 Image acquisition and hardware synchronization are realized by custom software. The open source software Micromanager can also be used to control the optical setup and acquisition.</li>
</ol>
4. Optical imaging
<ol>
	<li>Place the stereotaxic frame under the stereomicroscope, the cranial window centered in the field of view (see Fig. 2A for a schematic of the optical setup).</li>
	<li>Tune the microscope to focus on the capillaries. Prior to stimulation trials (see description in 5) an image of the bulb is taken under 560nm (green) light (fiber ring) which provides a good contrast for blood vessels imaging. This picture is used as an anatomic control to check the state of the preparation and is acquired several times during the experiment.</li>
	<li>For IOS imaging, reflected light intensity is recorded by the CCD camera under 630+/-10 nm (red) illumination. Images are acquired at full frame (no binning) at 5 frames per second which correspond to an exposure time of about 150ms. Power of the light source is adjusted to reach gray levels of at least &tilde;3000 on the OB area, close to the saturation of the CCD pixels wells. Doing so makes it possible to take advantage of the 12bits dynamics of the CCD to capture faint intensity changes during activation. Maximum IOS amplitudes reach &tilde;1%.</li>
	<li>For FAS imaging fluorescence is acquired under excitation of 480nm+/-20nm (blue) epiflurorescence light. A high-pass filter at 515nm is set up to capture light. Images are acquired at 5 frames per second with a binning of 4 by 4 to maximize sensitivity. Excitation light power is adjusted similarly to IOS with gray levels of 3000 on the OB area. Maximum FAS amplitudes reach &tilde;3%.
	 For both imaging modalities, the depth of field in the subject plane is the same and was measured at 0.5 mm for a magnification of about 4 times.</li>
	<li>Light should be turned off between imaging trials to avoid heating and photobleaching of the preparation.</li>
</ol>
5. Imaging trials
<ol>
	<li>Standard imaging session included a baseline of 5 to 10s where only air is delivered, followed by the odor stimulation for 3 to 10s depending on the chosen odor concentration, and 70 to 82s are further recorded with a constant air flow for baseline recovery (Fig. 2A). At the end of the trial, light is turned off and pure air is delivered for the inter trial interval duration (1 to 3 minutes) to wash out residual odor molecules and avoid sensory habituation. Odor trials were interleaved with blank trials (air delivery).</li>
	<li>Odor-evoked activated zones are visualized as roughly spherical areas on the maps and correspond to the size of individual glomeruli (80 to 200&mu;m in diameter9). Image processing is explained in Figure 2B. Olfactory maps are presented in Figure 3. Contrary to any other sensory system, the signal-to-noise ratio in the OB was sufficient to resolve odor-evoked responses in single trials (Fig. 3B for IOS and Fig. 3D for FAS).</li>
	<li>Mice are euthanized immediately at the end of the imaging session using methods in accordance with European recommendations for care and use of laboratory animals.</li>
</ol>
6. Representative Results (see olfactory maps in figure 3):
<img alt="Figure 1" src="/files/ftp_upload/3336/3336fig1.jpg" /> 
Figure 1 Structural organization of the main olfactory bulb in rodents. Olfactory sensory neurons, primary sensory cells located in the main olfactory epithelium, express the same odorant receptor and converge onto the same glomeruli in the OB. Olfactory glomeruli, the round-shaped neuropils (dashed circles), are located at the surface of the OB. Note that a very dense and complex vascular network is present at the glomerular level. Abbreviations (top/down): ONL: olfactory nerve layer; GL: glomerular layer, EPL: external plexiform layer; MCL: mitral cell layer; GCL: granule cell layer.
<img alt="Figure 2" src="/files/ftp_upload/3336/3336fig2.jpg" /> 
Figure 2 Reflectance and fluorescence signals recording in vivo. A. Wide-field optical imaging setup. The brain of an anesthetized mouse is exposed to either red (IOS) or blue (FAS) light through either an annular fiber ring attached to the optics lens or an epi-illumination port of a microscope. Odors are loaded into sealed vials and odorized air is delivered to the animal nose (green light: open valve). B. Recording protocol and data processing. IOS and FAS are recorded as series of individual trials (90s of duration). The diagram shows the timeline of a single trial: baseline varies from 5 to 10s, stimulation from 3 to 10s, and return to baseline from 70 to 82s. Image processing requires pixel-by-pixel subtraction of intensity values during the baseline to intensity values during periods of stimulation (for FAS) or stimulation plus return to baseline (for IOS). This difference is then divided by baseline values to obtain a variation in % (see resulting images in Fig. 3).
<img alt="Figure 3" src="/files/ftp_upload/3336/3336fig3.jpg" /> 
Figure 3 Odor-evoked activity maps in the OB using IOS and FAS imaging. A. Vasculature of the dorsal OB visualized under green light. B-C. IOS imaged (single trial versus three averaged trials respectively) for a 10s presentation of 20% hexanal. White arrows indicate the spherical regions of interest activated by this odor. These activation maps were obtained using the frames averaged during the first second after the end of odor stimulation (maximum of reflectance variation -0.63% in A and -0.52% in B). Note the black zones of absorbance where odor activation has occurred. C-D. FAS acquired sequentially in the same mouse for the same odorant (single trial versus three averaged trials respectively). These activation maps were obtained using the frames averaged during the first second after the beginning of odor stimulation (maximum of fluorescence variation +0.72% in D and +0.66% in E). Note that the white zones of autofluorescence emission indicated by black arrows correspond to the black zones in IOS. The grainy aspect seen in the FAS map is due to the 4 by 4 binning required for the improvement of the sensitivity. FAS images were not corrected from autofluorescence bleaching. Actual dimensions of images B-E: 0.7 mm wide x 1.2 mm long.

<ol>
	<li>Grinvald, A., Lieke, E., Frostig, R. D., Gilbert, C. D., Wiesel, T. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+architecture+of+cortex+revealed+by+optical+imaging+of+intrinsic+signals.">Functional architecture of cortex revealed by optical imaging of intrinsic signals.</a> Nature. 324, 361-364 (1986).</li><li>Rubin, B. D., Katz, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+odorant+representations+in+the+mammalian+olfactory+bulb.">Optical imaging of odorant representations in the mammalian olfactory bulb.</a> Neuron. 23, 499-511 (1999).</li><li>Chance, B., Cohen, P., J&amp;246, b. s. i. s., &amp;amp, F., Schoener, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+oxidation-reduction+states+in+vivo.">Intracellular oxidation-reduction states in vivo.</a> Science. 137, 499-508 (1962).</li><li>Shibuki, K., Hishida, R., Murakami, H., Kudoh, M., Kawaguchi, T., Watanabe, M., Watanabe, S., Kouuchi, T., Tanaka, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+imaging+of+somatosensory+cortical+activity+in+the+rat+visualized+by+flavoprotein+autofluorescence.">Dynamic imaging of somatosensory cortical activity in the rat visualized by flavoprotein autofluorescence.</a> J. Physiol. 549 (Pt. 3, 919-9127 (2003).</li><li>Uchida, N., Takahashi, Y. K., Tanifuji, M., Mori, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odor+maps+in+the+mammalian+olfactory+bulb:+domain+organization+and+odorant+structural+features.">Odor maps in the mammalian olfactory bulb: domain organization and odorant structural features.</a> Nat. Neurosci. 3, 1035-1043 (2000).</li><li>Gurden, H., Uchida, N., Mainen, Z. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory-evoked+intrinsic+optical+signals+in+the+olfactory+bulb+are+coupled+to+glutamate+release+and+uptake.">Sensory-evoked intrinsic optical signals in the olfactory bulb are coupled to glutamate release and uptake.</a> Neuron. 52, 335-345 (2006).</li><li>Soucy, E. R., Albeanu, D. F., Fantana, A. L., Murthy, V. N., Meister, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precision+and+diversity+in+an+odor+map+on+the+olfactory+bulb.">Precision and diversity in an odor map on the olfactory bulb.</a> Nat. Neurosci. 12, 210-220 (2009).</li><li>Frostig, R. D., Lieke, E. E., Ts'o, D. Y., Grinvald, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+functional+architecture+and+local+coupling+between+neuronal+activity+and+the+microcirculation+revealed+by+in+vivo+high-resolution+optical+imaging+of+intrinsic+signals.">Cortical functional architecture and local coupling between neuronal activity and the microcirculation revealed by in vivo high-resolution optical imaging of intrinsic signals.</a> Proc. Natl. Acad. Sci. U.S.A. 87, 6082-6086 (1990).</li><li>Meister, M., Bonhoeffer, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuning+and+topography+in+an+odor+map+on+the+rat+olfactory+bulb.">Tuning and topography in an odor map on the rat olfactory bulb.</a> J. Neurosci. 21, 1351-1360 (2001).</li><li>Dunn, A. K., Devor, A., Bolay, H., Andermann, M. L., Moskowitz, M. A., Dale, 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=Simultaneous+imaging+of+total+cerebral+hemoglobin+concentration,+oxygenation,+and+blood+flow+during+functional+activation.">Simultaneous imaging of total cerebral hemoglobin concentration, oxygenation, and blood flow during functional activation.</a> Opt. Lett. 28, 28-30 (2003).</li><li>L'Heureux, B., Gurden, H., Pain, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autofluorescence+imaging+of+NADH+and+flavoproteins+in+the+rat+brain:+insights+from+Monte+Carlo+simulations.">Autofluorescence imaging of NADH and flavoproteins in the rat brain: insights from Monte Carlo simulations.</a> Optics. Express. 17, 9477-9490 (2009).</li><li>Renaud, R., Gurden, H., Chery, R., Bendhamane, M., Martin, C., Pain, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multispectral+imaging+of+the+olfactory+bulb+activation:+influence+of+realistic+differential+pathlength+correction+factors+on+the+derivation+of+oxygenation+and+total+hemoglobin+concentration+maps+(Proceedings+Paper).">Multispectral imaging of the olfactory bulb activation: influence of realistic differential pathlength correction factors on the derivation of oxygenation and total hemoglobin concentration maps (Proceedings Paper).</a> , (2011).</li></ol>

We have nothing to disclose.

In this article we present IOS and FAS imaging techniques for in vivo recordings of odor-evoked activities in the mouse OB. To achieve this goal a relatively simple and affordable wide-field optical imaging setup is necessary. The acquisition of imaging data requires training to perform the fine surgical procedures and avoid any injury to the dura or brain tissue. In particular, major bleeding will absorb photons recorded for imaging and end up the experiment.
One benefit of IOS and FAS imaging is to avoid the injection of fluorescent tracers that could result in cellular toxicity or undesired side effects. They make it possible to tackle issues about olfactory maps thus spatial coding of sensory stimuli. Contrary to 2-DeoxyGlucose imaging, they provide the possibility to image several odors in a single animal. However, since photon penetration is limited in the tissue, IOS and FAS are restricted to the dorsal part of the OB and can not be recorded from ventral regions.
Endogenous optical signal imaging offers excellent spatial resolution for in vivo imaging. Technical improvements concern quantitative calculations of vascular components in the reflectance signals8,9 as well as dynamics of blood oxygenation and volume during sensory activation10. Multiwavelength imaging of IOS imaging approaches are currently developed in our laboratory to fully quantify total hemoglobin concentration and oxygenation in the OB during sensory activation. These spectroscopic optical measurements added to FAS imaging will give the opportunity to answer the unsolved relationship between vascular and intracellular dynamics during sensory activation11,12.

<table border="1">
<tbody>
<tr>
 <td>Name of The Regent</td>
	 <td>Company</td>
	 <td>Catalogue number</td>
</tr>
<tr>
 <td>Imalgene</td>
	 <td>Merial</td>
	 <td></td>
</tr>
<tr>
 <td>Rompun</td>
	 <td>Bayer</td>
	 <td></td>
</tr>
<tr>
 <td>Agarose, type III-A</td>
	 <td>Sigma-Aldrich</td>
	 <td>A9793-50G</td>
</tr>
<tr>
 <td>Hexanal 98%</td>
	 <td>Aldrich</td>
	 <td>115606-100ML</td>
</tr>
</tbody>
</table>

imaging odor-evoked activities in the mouse olfactory bulb using optical reflectance and autofluorescence signals

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. Functional optical imaging techniques are advantageous to visualize the activation of these modules in sensory cortices with high spatial resolution. In this context, endogenous optical signals that arise from molecular mechanisms linked to neuroenergetics are valuable sources of contrast to record spatial maps of sensory stimuli over wide fields in the rodent brain.
Here, we present two techniques based on changes of endogenous optical properties of the brain tissue during activation. First the intrinsic optical signals (IOS) are produced by a local alteration in red light reflectance due to: (i) absorption by changes in blood oxygenation level and blood volume (ii) photon scattering. The use of in vivo IOS to record spatial maps started in the mid 1980's with the observation of optical maps of whisker barrels in the rat and the orientation columns in the cat visual cortex1. IOS imaging of the surface of the rodent main olfactory bulb (OB) in response to odorants was later demonstrated by Larry Katz's group2. The second approach relies on flavoprotein autofluorescence signals (FAS) due to changes in the redox state of these mitochondrial metabolic intermediates. More precisely, the technique is based on the green fluorescence due to oxidized state of flavoproteins when the tissue is excited with blue light. Although such signals were probably among the first fluorescent molecules recorded for the study of brain activity by the pioneer studies of Britton Chances and colleagues3, it was not until recently that they have been used for mapping of brain activation in vivo. FAS imaging was first applied to the somatosensory cortex in rodents in response to hindpaw stimulation by Katsuei Shibuki's group4.
The olfactory system is of central importance for the survival of the vast majority of living species because it allows efficient detection and identification of chemical substances in the environment (food, predators). The OB is the first relay of olfactory information processing in the brain. It receives afferent projections from the olfactory primary sensory neurons that detect volatile odorant molecules. Each sensory neuron expresses only one type of odorant receptor and neurons carrying the same type of receptor send their nerve processes to the same well-defined microregions of &tilde;100&mu;m3 constituted of discrete neuropil, the olfactory glomerulus (Fig. 1). In the last decade, IOS imaging has fostered the functional exploration of the OB5, 6, 7 which has become one of the most studied sensory structures. The mapping of OB activity with FAS imaging has not been performed yet.
Here, we show the successive steps of an efficient protocol for IOS and FAS imaging to map odor-evoked activities in the mouse OB.

Watch this Scientific Journal Video about Imaging Odor-Evoked Activities in the Mouse Olfactory Bulb using Optical Reflectance and Autofluorescence Signals at JoVE.com

Imaging Odor-Evoked Activities in the Mouse Olfactory Bulb using Optical Reflectance and Autofluorescence Signals

This article presents the protocols of intrinsic optical signals and flavoproteins autofluorescence signals imaging to map odor-evoked activities at the surface of the olfactory bulb in mice.

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. ...

imaging-odor-evoked-activities-mouse-olfactory-bulb-using-optical

Research

JoVE Journal

Neuroscience

16.0K 조회수.  UMR8165 Université Paris Sud 11, Paris Diderot 7 – CNRS. 이 문서는 생쥐의 후각 망울의 표면에지도 냄새 evoked 활동에 고유 광 신호와 flavoproteins autofluorescence 신호 이미징의 프로토콜을 제공합니다.

비디오: Imaging Odor-Evoked Activities in the Mouse Olfactory Bulb using Optical Reflectance and Autofluorescence Signals

Analyzing Responses of Mouse Olfactory Sensory Neurons Using the Air-phase Electroolfactogram Recording

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and other social interactions. The electroolfactogram (EOG) recording is an informative, easy to conduct, and reliable method to assay olfactory function at the level of the olfactory epithelium. Since the 1956 description of the EOG by Ottoson in frogs1, the EOG recording has been applied in many vertebrates including salamanders, rabbits, rats, mice, and humans (reviewed by Scott and Scott-Johnson, 2002, ref. 2). The recent advances in genetic modification in mice have rekindled interest in recording the EOG for physiological characterization of olfactory function in knock-out and knock-in mice. EOG recordings have been successfully applied to demonstrate the central role of olfactory signal transduction components3-8, and more recently to characterize the contribution of certain regulatory mechanisms to OSN responses9-12.
Odorant detection occurs at the surface of the olfactory epithelium on the cilia of OSNs, where a signal transduction cascade leads to opening of ion channels, generating a current that flows into the cilia and depolarizes the membrane13. The EOG is the negative potential recorded extracellularly at the surface of the olfactory epithelium upon odorant stimulation, resulting from a summation of the potential changes caused by individual responsive OSNs in the recording field2. Comparison of the amplitude and kinetics of the EOG thus provide valuable information about how genetic modification and other experimental manipulations influence the molecular signaling underlying the OSN response to odor. 
Here we describe an air-phase EOG recording on a preparation of mouse olfactory turbinates. Briefly, after sacrificing the mouse, the olfactory turbinates are exposed by bisecting the head along the midline and removing the septum. The turbinate preparation is then placed in the recording setup, and a recording electrode is placed at the surface of the olfactory epithelium on one of the medial turbinates. A reference electrode is electrically connected to the tissue through a buffer solution. A continuous stream of humidified air is blown over the surface of the epithelium to keep it moist. The vapor of odorant solutions is puffed into the stream of humidified air to stimulate the epithelium. Responses are recorded and digitized for further analysis.

The electroolfactogram (EOG) recording is an informative, easy-to-conduct, and reliable way of assessing olfactory function at the level of the olfactory epithelium. This protocol describes a recording setup, mouse tissue preparation, data collection, and basic data analysis.

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and ...

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

In vivo electroporation is a powerful method for delivering DNA expression plasmids, RNAi reagents, and morpholino anti-sense oligonucleotides to specific regions of developing embryos, including those of C. elegans, chick, Xenopus, zebrafish, and mouse 1. In zebrafish, in vivo electroporation has been shown to have excellent spatial and temporal resolution for the delivery of these reagents 2-7. The temporal resolution of this method is important because it allows for incorporation of these reagents at specific stages in development. Furthermore, because expression from electroporated vectors occurs within 6 hours 7, this method is more timely than transgenic approaches. While the spatial resolution can be extremely precise when targeting a single cell 2, 6, it is often preferable to incorporate reagents into a specific cell population within a tissue or structure. When targeting multiple cells, in vivo electroporation is efficient for delivery to a specific region of the embryo; however, particularly within the developing nervous system, it is difficult to target specific cell types solely through spatially discrete electroporation. Alternatively, enhancer trap transgenic lines offer excellent cell type-specific expression of transgenes 8. Here we describe an approach that combines transgenic Gal4-based enhancer trap lines 8 with spatially discrete in vivo electroporation 7, 9 to specifically target developing neurons of the zebrafish olfactory bulb. The Et(zic4:Gal4TA4,UAS:mCherry)hzm5 (formerly GA80_9) enhancer trap line previously described 8, displays targeted transgenic expression of mCherry mediated by a zebrafish optimized Gal4 (KalTA4) transcriptional activator in multiple regions of the developing brain including hindbrain, cerebellum, forebrain, and the olfactory bulb. To target GFP expression specifically to the olfactory bulb, a plasmid with the coding sequence of GFP under control of multiple Gal4 binding sites (UAS) was electroporated into the anterior end of the forebrain at 24-28 hours post-fertilization (hpf). Although this method incorporates plasmid DNA into multiple regions of the forebrain, GFP expression is only induced in cells transgenically expressing the KalTA4 transcription factor. Thus, by using the GA080_9 transgenic line, this approach led to GFP expression exclusively in the developing olfactory bulb. GFP expressing cells targeted through this approach showed typical axonal projections, as previously described for mitral cells of the olfactory bulb 10. This method could also be used for targeted delivery of other reagents including short-hairpin RNA interference expression plasmids, which would provide a method for spatially and temporally discrete loss-of-function analysis.

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

The temporal and spatial resolution of genetic manipulations determines the spectrum of biological phenomena that they can perturb. Here we use temporally and spatially discrete in vivo electroporation, combined with transgenic lines of zebrafish, to induce expression of a GFP transgene specifically in neurons of the developing olfactory bulb.

In vivo electroporation is a powerful method for delivering DNA expression plasmids, RNAi reagents, and morpholino anti-sense oligonucleotides to specific ...

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory bulb1-5. Olfactory information in the external world is transmitted to the antennal lobe by olfactory sensory neurons (OSNs), which segregate to distinct regions of neuropil called glomeruli according to the specific olfactory receptor they express. Here, OSN axons synapse with both local interneurons (LNs), whose processes can innervate many different glomeruli, and projection neurons (PNs), which convey olfactory information to higher olfactory brain regions.
Optical imaging of the activity of OSNs, LNs and PNs in the antennal lobe - traditionally using synthetic calcium indicators (e.g. calcium green, FURA-2) or voltage-sensitive dyes (e.g. RH414) - has long been an important technique to understand how olfactory stimuli are represented as spatial and temporal patterns of glomerular activity in many species of insects6-10. Development of genetically-encoded neural activity reporters, such as the fluorescent calcium indicators G-CaMP11,12 and Cameleon13, the bioluminescent calcium indicator GFP-aequorin14,15, or a reporter of synaptic transmission, synapto-pHluorin16 has made the olfactory system of the fruitfly, Drosophila melanogaster, particularly accessible to neurophysiological imaging, complementing its comprehensively-described molecular, electrophysiological and neuroanatomical properties2,4,17. These reporters can be selectively expressed via binary transcriptional control systems (e.g. GAL4/UAS18, LexA/LexAop19,20, Q system21) in defined populations of neurons within the olfactory circuitry to dissect with high spatial and temporal resolution how odor-evoked neural activity is represented, modulated and transformed22-24.
Here we describe the preparation and analysis methods to measure odor-evoked responses in the Drosophila antennal lobe using G-CaMP25-27. The animal preparation is minimally invasive and can be adapted to imaging using wide-field fluorescence, confocal and two-photon microscopes.

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

We describe an established technique to measure and analyze odor-evoked calcium responses in the antennal lobe of living Drosophila melanogaster.

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the &#947;-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the &#947;-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb.

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose.

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system. This system is constructed from a broadband white light source, a motorized filter wheel with a set of narrowband interference filters, a light guide, a collecting lens, a video zoom lens, and a monochromatic charged-coupled device (CCD) camera. An ellipsoidal cranial window is made in the skull bone of a rat under isoflurane anesthesia to capture in vivo multispectral diffuse reflectance images of the cortical surface. Regulation of the fraction of inspired oxygen using a gas mixture device enables the induction of different respiratory states such as normoxia, hyperoxia, and anoxia. A Monte Carlo simulation-based multiple regression analysis for the measured multispectral diffuse reflectance images at nine wavelengths (500, 520, 540, 560, 570, 580, 600, 730, and 760 nm) is then performed to visualize the two-dimensional maps of hemodynamics and the light scattering properties of the in vivo rat brain.

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system.

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory Sensory Neuron (OSN) is a bipolar cell with a single dendrite and a single unbranched axon. An OSN expresses only one Olfactory Receptor (OR) gene, OSNs expressing a given type of OR converge their axons to a few sets of invariant glomeruli in the Olfactory Bulb (OB). A remarkable feature of OSN projection is that the expressed ORs play instructive roles in axonal projection. ORs regulate the expression of multiple axon-sorting molecules and generate the combinatorial molecular code of axon-sorting molecules at the OSN axon termini. Thus, to understand the molecular mechanisms of OR-specific axon guidance mechanisms, it is vital to characterize their expression profiles at the OSN axon termini within the same glomerulus. The aim of this article was to introduce methods for collecting as many glomeruli as possible on a single OB section and for performing immunostaining using multiple antibodies. This would allow the comparison and analysis of the expression patterns of axon-sorting molecules without staining variation between OB sections.

Olfactory sensory neurons express a wide variety of axon-sorting molecules to establish proper neural circuitry. This protocol describes an immunohistochemical staining method to visualize combinatorial expressions of axon-sorting molecules at the axon termini of olfactory sensory neurons.

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory ...

Stereological Estimation of Dopaminergic Neuron Number in the Mouse Substantia Nigra Using the Optical Fractionator and Standard Microscopy Equipment

In pre-clinical Parkinson&#39;s disease research, analysis of the nigrostriatal tract, including quantification of dopaminergic neuron loss within the substantia nigra, is essential. To estimate the total dopaminergic neuron number, unbiased stereology using the optical fractionator method is currently considered the gold standard. Because the theory behind the optical fractionator method is complex and because stereology is difficult to achieve without specialized equipment, several commercially available complete stereology systems that include the necessary software do exist, purely for cell counting reasons. Since purchasing a specialized stereology setup is not always feasible, for many reasons, this report describes a method for the stereological estimation of dopaminergic neuronal cell counts using standard microscopy equipment, including a light microscope, a motorized object table (x, y, z plane) with imaging software, and a computer for analysis. A step-by-step explanation is given on how to perform stereological quantification using the optical fractionator method, and pre-programmed files for the calculation of estimated cell counts are provided. To assess the accuracy of this method, a comparison to data obtained from a commercially available stereology apparatus was performed. Comparable cell numbers were found using this protocol and the stereology device, thus demonstrating the precision of this protocol for unbiased stereology.

This work presents a step-by-step protocol for the unbiased stereological estimation of dopaminergic neuronal cell numbers in the mouse substantia nigra using standard microscopy equipment (i.e., a light microscope, a motorized object table (x, y, z plane), and public domain software for digital image analysis.

In pre-clinical Parkinson&#39;s disease research, analysis of the nigrostriatal tract, including quantification of dopaminergic neuron loss within the ...