This work was supported by the Mitsubishi Foundation, the Takeda Science Foundation, JST PRESTO and JSPS KAKENHI Grant Number 16H06144.

All experimental procedures were performed with the approval of the animal experiment ethics committee at the University of Tokyo and according to the University of Tokyo guidelines for the care and use of laboratory animals.
1. Preparation of Solutions
<ol>
	<li>Prepare 0.01 M Phosphate-Buffered Saline (PBS): add a PBS tablet (0.14 M NaCl, 0.0027 M KCl, 0.010 M PO43-, pH 7.4) to 1 L distilled water and stir at RT until it is completely dissolved.</li>
	<li>Prepare 4% paraformaldehyde (PFA) in PBS: add 4 g of PFA to 100 mL PBS. Stir the solution at 60 &#176;C until it is completely dissolved.
	<ol>
		<li>After dissolving the PFA in PBS, adjust the pH of the solution to 7.4 with 1 N sodium hydroxide (NaOH). Then, cool on ice and filter the solution through a filter paper to remove any particulate matter. Store at 4 &#176;C. 
		Caution: Take appropriate care when using these reagents. Handle with care and use gloves, safety goggles, and lab coat under a chemical hood. 
		NOTE: Use fresh 4% PFA solution prepared within 2 d.</li>
	</ol>
	</li>
	<li>Prepare 0.01 M PBS supplemented with 0.3% Triton X-100 (PBST): add 3 mL of Triton X- 100 to 997 mL of PBS. Stir the solution at RT until it is completely dissolved.</li>
	<li>Prepare 1% and 5% blocking solution: add 10 g of skim milk to 200 mL PBST to prepare 5% blocking solution. Stir at RT until it is completely dissolved. Dilute 5% blocking solution five times with PBST to prepare 1% blocking solution.</li>
</ol>
2. Preparation of Parasagittal OB Sections
<ol>
	<li>Use animals between 1-2 weeks of age. These age groups are suitable for the following experiments because glomeruli are clearly visible and the brain can be cut with the skull.</li>
	<li>Anesthetize the animal with an intraperitoneal injection of pentobarbital (50 mg/kg body weight). Perfuse the animal transcardially with ice-cold 4% PFA in PBS. Handle with care and use gloves, safety goggles, and lab coat under a chemical hood.</li>
	<li>After perfusion, cut off the head with scissors and remove the skin carefully. Then, insert a blade of the scissors into the space between the upper and lower teeth and cut horizontally to remove the lower jawbone. Trim away excess tissue with forceps and scissors to leave the olfactory tissue including the OB and OE. 
	NOTE: Do not remove the skull surrounding the OB as this retains the medial glomeruli aligned vertically straight.</li>
	<li>Immerse the olfactory tissue in PBS to remove the air in the nasal cavity. Cut off the posterior part of the brain vertically and place the olfactory tissue on the bottom of the embedding mold with the cutting face down. Fill the mold with Optimal Cutting Temperature (O.C.T.) compound and then immerse the mold into liquid nitrogen.
	<ol>
		<li>After the tissue in the compound is completely frozen, incubate it in the cryostat at -20 &#176;C for 1 h.</li>
	</ol>
	</li>
	<li>Make serial parasagittal sections (10 &#181;m) of the OB with a cryostat and collect them by sticking to MAS-coated glass slides. After sticking, dry the slides immediately with a blow dryer.</li>
</ol>
3. Day 1:&#160;Quadruple Immunostaining of the OB Slices&#160;
<ol>
	<li>Wash the slides for 5 min with PBS at RT. Repeat 3 times.</li>
	<li>Block the nonspecific binding sites by incubating the slides for 1 h with the 5% blocking solution at RT.</li>
	<li>Incubate the slides O/N with a cocktail of primary antibodies (400 &#181;L each slide) in the 1% blocking solution at RT. Use the following primary antibodies: guinea pig anti-Kirrel2 antibody (1:1000), goat anti-Semaphorin-7A (Sema7A) antibody (1:500), rat anti-OL-protocadherin (OLPC) antibody (1:500), and mouse anti-vesicular glutamate transporter2 (VGLUT2) antibody (1:500).</li>
</ol>
4. Day 2:&#160;Quadruple Immunostaining of the OB Slices&#160;
<ol>
	<li>Discard the primary antibody solution and wash the slides for 5 min with PBST at RT. Repeat 3 times.</li>
	<li>Incubate the slides for 1 h with a cocktail of secondary antibodies (400 &#181;L each slide) in PBS at RT. Use the following secondary antibodies: donkey anti-mouse Alexa Fluor 405 (1:400), donkey anti-goat Alexa Fluor 488 (1:400), donkey anti-guinea pig Alexa Fluor 555 (1:400), and donkey anti-rat Alexa Fluor 647 (1:400). 
	NOTE: All secondary antibodies should come from the same host species. Choose a different wavelength of fluorescence of secondary antibodies for each primary antibody to ensure no overlap of wavelengths.</li>
	<li>Discard the solution and wash the slides for 5 min with PBS at RT. Repeat 3 times.</li>
	<li>Mount coverslips on the slides with the mounting medium. For this, apply 2 drops of the mounting medium on each slide, and then, put a coverslip by removing the air bubbles.</li>
</ol>
5. Intensity Measurements
<ol>
	<li>Obtain fluorescent images with a fluorescence microscope. Use the following filter cubes: DAPI filter cube (360/40 nm excitation, 460/50 nm emission, and 400 nm dichroic mirror) for Alexa Fluor 405 signals, GFP filter cube (470/40 nm excitation, 525/50 nm emission, and 495 nm dichroic mirror) for Alexa Fluor 488, TRITC filter cube (545/25 nm excitation, 605/70 nm emission, and 565 nm dichroic mirror) for Alexa Fluor 555, and Cy5 filter cube (620/60 nm excitation, 700/75 nm emission, and 600 nm dichroic mirror) for Alexa Fluor 647. 
	Note: Adjust the exposure time to obtain fluorescent signals of the image without saturation.</li>
	<li>Define the glomerular structure by immunofluorescence signals of VGLUT2. Measure staining intensities of axon-sorting molecules within glomeruli with ImageJ.</li>
</ol>
6. Data Analysis
<ol>
	<li>Subtract background signals from the staining intensities of axon-sorting molecules.</li>
	<li>Prepare the expression data matrix, which has columns composed of axon-sorting molecules and rows composed of the glomeruli.</li>
	<li>Perform a principal component analysis (PCA) using the prepared expression dataset. Then, obtain the PCA scores, contribution ratios, and factor loadings of each principal component.</li>
</ol>

<ol>
	<li>Mori, K., Sakano, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+is+the+olfactory+map+formed+and+interpreted+in+the+mammalian+brain?.">How is the olfactory map formed and interpreted in the mammalian brain?.</a> Annu Rev Neurosci. 34, 467-499 (2011).</li><li>Takeuchi, H., Sakano, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+map+formation+in+the+mouse+olfactory+system.">Neural map formation in the mouse olfactory system.</a> Cell Mol Life Sci. 71, 3049-3057 (2014).</li><li>Mombaerts, 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=Visualizing+an+olfactory+sensory+map.">Visualizing an olfactory sensory map.</a> Cell. 87, 675-686 (1996).</li><li>Feinstein, P., Mombaerts, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+contextual+model+for+axonal+sorting+into+glomeruli+in+the+mouse+olfactory+system.">A contextual model for axonal sorting into glomeruli in the mouse olfactory system.</a> Cell. 117, 817-831 (2004).</li><li>Ishii, 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=Monoallelic+expression+of+the+odourant+receptor+gene+and+axonal+projection+of+olfactory+sensory+neurones.">Monoallelic expression of the odourant receptor gene and axonal projection of olfactory sensory neurones.</a> Genes Cell. 6, 71-78 (2001).</li><li>Nakashima, A., 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=Agonist-independent+GPCR+activity+regulates+anterior-posterior+targeting+of+olfactory+sensory+neurons.">Agonist-independent GPCR activity regulates anterior-posterior targeting of olfactory sensory neurons.</a> Cell. 154, 1314-1325 (2013).</li><li>Serizawa, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+neuronal+identity+code+for+the+odorant+receptor-specific+and+activity-dependent+axon+sorting.">A neuronal identity code for the odorant receptor-specific and activity-dependent axon sorting.</a> Cell. 127, 1057-1069 (2006).</li><li>Kaneko-Goto, T., Yoshihara, S., Miyazaki, H., Yoshihara, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BIG-2+mediates+olfactory+axon+convergence+to+target+glomeruli.">BIG-2 mediates olfactory axon convergence to target glomeruli.</a> Neuron. 57, 834-846 (2008).</li><li>Ihara, N., Nakashima, A., Hoshina, N., Ikegaya, Y., Takeuchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+axon-sorting+molecules+in+mouse+olfactory+sensory+neurons.">Differential expression of axon-sorting molecules in mouse olfactory sensory neurons.</a> Eur J Neuro. 44, 1998-2003 (2016).</li><li>Williams, E. O., 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=Delta+Protocadherin+10+is+Regulated+by+Activity+in+the+Mouse+Main+Olfactory+System.">Delta Protocadherin 10 is Regulated by Activity in the Mouse Main Olfactory System.</a> Front Neural Circuits. 5, 9 (2011).</li><li>Brunet, L. J., Gold, G. H., Ngai, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+anosmia+caused+by+a+targeted+disruption+of+the+mouse+olfactory+cyclic+nucleotide-gated+cation+channel.">General anosmia caused by a targeted disruption of the mouse olfactory cyclic nucleotide-gated cation channel.</a> Neuron. 17, 681-693 (1996).</li></ol>

The authors declare no competing financial interests.

Quadruple immunostaining of parasagittal OB sections enabled the visualization and quantification of the expression levels of as many as four axon-sorting molecules simultaneously in a larger number of glomeruli. By analyzing these multivariable data with PCA, the characteristics for the expression of those molecules can be speculated.
For successful staining, the tissue sample preparation is critically important. Some protocols suggest that tissues should be post-fixed with 4% PFA and treated...

During development, neurons are precisely connected with each other to form proper neural circuits, which is critical for the normal brain function. Since aberrant neural circuits in the brain are thought to be the cause of mental disorders such as autism and schizophrenia, understanding the mechanisms of neural circuit formation is one of the major challenges in the field of neuroscience.
In the mouse olfactory system, each Olfactory Sensory Neuron (OSN) in the Olfactory Epithelium (OE) expresses only one functional Olfactory Receptor (OR) gene and OSNs expressing the same OR converge their axons to a specific pair of glomeruli at stereotyped locations in the Olfactory Bulb (OB)1,2. The mouse olfactory system is an excellent model system for studying the molecular mechanisms of neural circuit formation because researchers can utilize the OR expression to identify a specific subtype of OSNs and visualize the projection sites of OSN axons as clear glomerular structures. A remarkable feature of OSN projection is that ORs play instructive roles in projecting OSN axons to the OB3,4,5,6. More specifically, after OSN axons are guided to approximate target regions, they are segregated to form glomerulus in an OR-dependent manner. Previous studies have shown that OR molecules control the expression of axon-sorting molecules, which regulate glomerular segregation7,8. Moreover, accumulating evidence suggests that OR molecules generate the neuronal identity code by a unique combination of axon-sorting molecules9. Thus, to understand the mechanism of OR-dependent glomerular segregation, it is necessary to characterize the expression profiles of axon-sorting molecules in OSNs.
Fluorescent immunostaining is a common method to visualize the expression of specific genes. Since proteins of axon-sorting molecules are predominantly localized to OSN axons, researchers need to use OB sections to characterize their expression patterns in OSNs. Coronal sectioning of the OB has been routinely used for immunostaining. However, this preparation loses the topographic information along the anterior-posterior axis in the same OB section. We therefore developed a parasagittal preparation of the medial side of the OB, which can mount as many surrounding glomeruli as possible on the same OB section. Combined with immunostaining using multiple antibodies, this preparation allows the comparison and analysis of the expression patterns of axon-sorting molecules without staining variation between OB sections.
Furthermore, an immunohistochemical staining method has been presented without post-fixation with PFA and sucrose treatment. This method allows researchers to obtain enough high-quality staining data for multivariable data analysis. The protocols presented here will provide details of powerful methods for researchers who study the olfactory neural circuit formation.

<table border="0" cellpadding="0" cellspacing="0" width="713">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">Phosphate Buffered Saline (PBS) Tablets, pH7.4</td>
			<td style="width:151px;">TAKARA BIO</td>
			<td style="width:119px;">T9181</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">Skim Milk</td>
			<td style="width:151px;">nacalai tesque</td>
			<td style="width:119px;">31149-75</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">goat anti-Sema7A antibody</td>
			<td style="width:151px;">R&#38;D Systems</td>
			<td style="width:119px;">AF2068</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">rat anti-OLPC antibody</td>
			<td style="width:151px;">Merck Millipore</td>
			<td style="width:119px;">MABT20</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">mouse anti-VGLUT2 antibody</td>
			<td style="width:151px;">Merck Millipore</td>
			<td style="width:119px;">MAB5504</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">goat anti-BIG-2 antibody</td>
			<td style="width:151px;">R&#38;D Systems</td>
			<td style="width:119px;">AF2205</td>
			<td></td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:277px;">gunea pig anti-Kirrel2 antibody</td>
			<td style="width:151px;">Operon Biotechnologies</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Anti-Kirrel2 antibodies were generated by immunizing guinea pigs with KLH-conjugated synthetic peptides (644-673aa): CRLYRARAGYLTTPHPRAFTSYMKPTSFGP</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">donkey anti-mouse Alexa Fluor 405</td>
			<td style="width:151px;">Abcam</td>
			<td style="width:119px;">ab175658</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:277px;">donkey anti-goat Alexa Fluor 488&#160;</td>
			<td style="width:151px;">Jackson ImmunoResearch</td>
			<td style="width:119px;">705-545-003</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:277px;">donkey anti-guinea pig Alexa Fluor 555</td>
			<td style="width:151px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">A21432</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:277px;">donkey anti-rat Alexa Fluor 647</td>
			<td style="width:151px;">Jackson ImmunoResearch</td>
			<td style="width:119px;">712-605-153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Paraformaldehyde</td>
			<td style="width:151px;">Wako</td>
			<td style="width:119px;">162-16065</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">MAS coated slide glasses</td>
			<td style="width:151px;">MATSUNAMI</td>
			<td style="width:119px;">MAS-01</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:277px;">forceps</td>
			<td style="width:151px;">Fine Science Tools</td>
			<td style="width:119px;">11253-27</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:277px;">Vannas Spring Scissors</td>
			<td style="width:151px;">Fine Science Tools</td>
			<td style="width:119px;">15000-00</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:277px;">dissecting scissors</td>
			<td style="width:151px;">Fine Science Tools</td>
			<td style="width:119px;">14090-09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">fluorescent microscope</td>
			<td style="width:151px;">KEYENCE</td>
			<td style="width:119px;">BZ-X700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">DAPI filter cube</td>
			<td style="width:151px;">KEYENCE</td>
			<td style="width:119px;">OP-87762</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">GFP filter cube</td>
			<td style="width:151px;">KEYENCE</td>
			<td style="width:119px;">OP-87763</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">TRITC filter cube</td>
			<td style="width:151px;">KEYENCE</td>
			<td style="width:119px;">OP-87764</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Cy5 filter cube</td>
			<td style="width:151px;">KEYENCE</td>
			<td style="width:119px;">OP-87766</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">filter paper</td>
			<td style="width:151px;">ADVANTEC</td>
			<td style="width:119px;">00011185</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">O.C.T compound</td>
			<td style="width:151px;">Sakura Finetek</td>
			<td style="width:119px;">M71484</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The olfactory glomerular map is formed by initial global targeting and subsequent glomerular segregation of OSN axons1,2. Glomerular segregation is regulated by the adhesive/repulsive axonal interactions mediated by axon-sorting molecules whose expression levels are determined by expressed OR molecules7. The axon-sorting molecules involved in glomerular segregation are expressed in a position-independent mo...

Watch this Scientific Journal Video about Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes at JoVE.com

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

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 ...

quadruple-immunostaining-olfactory-bulb-for-visualization-olfactory

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

7.0K Views.  The University of Tokyo. 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.

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

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 ...

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. 
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

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 ...

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 ...

Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory  Neurons

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. Caged compounds are molecules made physiologically inactive by a chemical cage that can be broken by a flash of ultraviolet light. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged compounds for the study of olfactory transduction in dissociated mouse olfactory sensory neurons. The process of olfactory transduction (Figure 1) takes place in the cilia of olfactory sensory neurons, where odorant binding to receptors leads to the increase of cAMP that opens cyclic nucleotide-gated (CNG) channels2. Ca entry through CNG channels activates Ca-activated Cl channels. We show how to dissociate neurons from the mouse olfactory epithelium3 and how to activate CNG channels or Ca-activated Cl channels by photolysis of caged cAMP4 or caged Ca5. We use a flash lamp6,7 to apply ultraviolet flashes to the ciliary region to uncage cAMP or Ca while patch-clamp recordings are taken to measure the current in the whole-cell voltage-clamp configuration8-11.

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged cAMP or caged Ca for the study of olfactory transduction in dissociated mouse olfactory sensory neurons.

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. ...

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.

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. ...

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps shape attention and also protects cells from over-stimulation. Adaptation within the olfactory circuit of C. elegans was first described by Colbert and Bargmann1,2. Here, the authors defined parameters of the olfactory adaptation paradigm, which they used to design a genetic screen to isolate mutants defective in their ability to adapt to volatile odors sensed by the Amphid Wing cells type C (AWC) sensory neurons. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor3 for 30 min they will adapt their responsiveness to the odor and will then ignore the adapting odor in a chemotaxis behavioral assay for ~1 hr. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor for ~1 hr they will then ignore the adapting odor in a chemotaxis behavioral assay for ~3 hr. These two phases of olfactory adaptation in C. elegans were described as short-term olfactory adaptation (induced after 30 min odor exposure), and long-term olfactory adaptation (induced after 60 min odor exposure). Later work from L'Etoile et al.,4 uncovered a Protein Kinase G (PKG) called EGL-4 that is required for both the short-term and long-term olfactory adaptation in AWC neurons. The EGL-4 protein contains a nuclear localization sequence that is necessary for long-term olfactory adaptation responses but dispensable for short-term olfactory adaptation responses in the AWC4. By tagging EGL-4 with a green fluorescent protein, it was possible to visualize the localization of EGL-4 in the AWC during prolonged odor exposure. Using this fully functional GFP-tagged EGL-4 (GFP::EGL-4) molecule we have been able to develop a molecular readout of long-term olfactory adaptation in the AWC5. Using this molecular readout of olfactory adaptation we have been able to perform both forward and reverse genetic screens to identify mutant animals that exhibit defective subcellular localization patterns of GFP::EGL-4 in the AWC6,7. Here we describe: 1) the construction of GFP::EGL-4 expressing animals; 2) the protocol for cultivation of animals for long-term odor-induced nuclear translocation assays; and 3) the scoring of the long-term odor-induced nuclear translocation event and recovery (re-sensitization) from the nuclear GFP::EGL-4 state.

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

Here we describe a molecular readout of long-term olfactory adaptation in Caenorhabditis elegans. The Protein Kinase G, EGL-4, is necessary for stable adaptation responses in the primary sensory neuron pair called AWC. During prolonged odor exposure EGL-4 translocates from the cytosol to nucleus of the AWC.

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Olfactory Assays for Mouse Models of Neurodegenerative Disease

In many neurodegenerative diseases and particularly in Parkinson&#8217;s disease, deficits in olfaction are reported to occur early in the disease process and may be a useful behavioral marker for early detection. Earlier detection in neurodegenerative disease is a major goal in the field because this is when neuroprotective therapies have the best potential to be effective. Therefore, in preclinical studies testing novel neuroprotective strategies in rodent models of neurodegenerative disease, olfactory assessment could be highly useful in determining therapeutic potential of compounds and translation to the clinic. In the present study we describe a battery of olfactory assays that are useful in measuring olfactory function in mice. The tests presented in this study were chosen because they measure olfaction abilities in mice related to food odors, social odors, and non-social odors. These tests have proven useful in characterizing novel genetic mouse models of Parkinson&#8217;s disease as well as in testing potential disease-modifying therapies.

Impairment in olfactory function is a common feature in many neurodegenerative disorders including Parkinson, Alzheimer, and Huntington diseases. In the present article, we describe a set of tests for assessing olfaction discrimination and detection in mice that can be used to measure olfactory abilities in mouse models of neurodegenerative diseases.

In many neurodegenerative diseases and particularly in Parkinson&#8217;s disease, deficits in olfaction are reported to occur early in the disease process ...

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 ...

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

Summary

Explore More Videos

Chapters in this video

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

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

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

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

Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory Neurons

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

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Olfactory Assays for Mouse Models of Neurodegenerative Disease

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