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/pubmed?term=((How+is+the+olfactory+map+formed+and+interpreted+in+the+mammalian+brain%3F%5BTitle%5D)+AND+%22Annu+Rev+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Neural+map+formation+in+the+mouse+olfactory+system%5BTitle%5D)+AND+%22Cell+Mol+Life+Sci%22%5BJournal%5D)">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/pubmed?term=((Visualizing+an+olfactory+sensory+map%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((A+contextual+model+for+axonal+sorting+into+glomeruli+in+the+mouse+olfactory+system%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((Monoallelic+expression+of+the+odourant+receptor+gene+and+axonal+projection+of+olfactory+sensory+neurones%5BTitle%5D)+AND+%22Genes+Cell%22%5BJournal%5D)">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/pubmed?term=((Agonist-independent+GPCR+activity+regulates+anterior-posterior+targeting+of+olfactory+sensory+neurons%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((A+neuronal+identity+code+for+the+odorant+receptor-specific+and+activity-dependent+axon+sorting%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((BIG-2+mediates+olfactory+axon+convergence+to+target+glomeruli%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">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/pubmed?term=((Differential+expression+of+axon-sorting+molecules+in+mouse+olfactory+sensory+neurons%5BTitle%5D)+AND+%22Eur+J+Neuro%22%5BJournal%5D)">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/pubmed?term=((Delta+Protocadherin+10+is+Regulated+by+Activity+in+the+Mouse+Main+Olfactory+System%5BTitle%5D)+AND+%22Front+Neural+Circuits%22%5BJournal%5D)">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/pubmed?term=((General+anosmia+caused+by+a+targeted+disruption+of+the+mouse+olfactory+cyclic+nucleotide-gated+cation+channel%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">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><tbody><tr><td>Phosphate-Buffered Saline (PBS) Tablets, pH7.4</td><td>TAKARA BIO</td><td>T9181</td><td></td></tr><tr><td>Skim Milk</td><td>nacalai tesque</td><td>31149-75</td><td></td></tr><tr><td>goat anti-Sema7A antibody</td><td>R&amp;amp;D Systems</td><td>AF2068</td><td></td></tr><tr><td>rat anti-OLPC antibody</td><td>Merck Millipore</td><td>MABT20</td><td></td></tr><tr><td>mouse anti-VGLUT2 antibody</td><td>Merck Millipore</td><td>MAB5504</td><td></td></tr><tr><td>goat anti-BIG-2 antibody</td><td>R&amp;amp;D Systems</td><td>AF2205</td><td></td></tr><tr><td>gunea pig anti-Kirrel2 antibody</td><td>Operon Biotechnologies</td><td></td><td>Anti-Kirrel2 antibodies were generated by immunizing guinea pigs with KLH-conjugated synthetic peptides (644-673aa): CRLYRARAGYLTTPHPRAFTSYMKPTSFGP</td></tr><tr><td>donkey anti-mouse Alexa Fluor 405</td><td>Abcam</td><td>ab175658</td><td></td></tr><tr><td>donkey anti-goat Alexa Fluor 488&amp;nbsp;</td><td>Jackson ImmunoResearch</td><td>705-545-003</td><td></td></tr><tr><td>donkey anti-guinea pig Alexa Fluor 555</td><td>Thermo Fisher Scientific</td><td>A21432</td><td></td></tr><tr><td>donkey anti-rat Alexa Fluor 647</td><td>Jackson ImmunoResearch</td><td>712-605-153</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Wako</td><td>162-16065</td><td></td></tr><tr><td>MAS coated slide glasses</td><td>MATSUNAMI</td><td>MAS-01</td><td></td></tr><tr><td>forceps</td><td>Fine Science Tools</td><td>11253-27</td><td></td></tr><tr><td>Vannas Spring Scissors</td><td>Fine Science Tools</td><td>15000-00</td><td></td></tr><tr><td>dissecting scissors</td><td>Fine Science Tools</td><td>14090-09</td><td></td></tr><tr><td>fluorescent microscope</td><td>KEYENCE</td><td>BZ-X700</td><td></td></tr><tr><td>DAPI filter cube</td><td>KEYENCE</td><td>OP-87762</td><td></td></tr><tr><td>GFP filter cube</td><td>KEYENCE</td><td>OP-87763</td><td></td></tr><tr><td>TRITC filter cube</td><td>KEYENCE</td><td>OP-87764</td><td></td></tr><tr><td>Cy5 filter cube</td><td>KEYENCE</td><td>OP-87766</td><td></td></tr><tr><td>filter paper</td><td>ADVANTEC</td><td>00011185</td><td></td></tr><tr><td>O.C.T compound</td><td>Sakura Finetek</td><td>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

Research

JoVE Journal

Neuroscience

7.1K Views.  The University of Tokyo.  Olfactorische zintuiglijke neuronen vormen een grote verscheidenheid aan axon-sorterende moleculen om een ​​goede neurale schakeling te vormen. Dit protocol beschrijft een immunohistochemische kleuring methode om combinatorische expressies van axon-sorterende moleculen te visualiseren bij de axon termini van olfactorische sensorische neuronen. 

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

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 ...

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. ...

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

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 ...

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

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

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

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

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

Whole Mount Labeling of Cilia in the Main Olfactory System of Mice

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single dendrite to the surface of the epithelium, ending in a structure called dendritic knob. Cilia emanate from this knob into the mucus covering the epithelial surface. The proteins of the olfactory signal transduction cascade are mainly localized in the ciliary membrane, being in direct contact with volatile substances in the environment. For a detailed understanding of olfactory signal transduction, one important aspect is the exact morphological analysis of signaling protein distribution. Using light microscopical approaches in conventional cryosections, protein localization in olfactory cilia is difficult to determine due to the density of ciliary structures. To overcome this problem, we optimized an approach for whole mount labeling of cilia, leading to improved visualization of their morphology and the distribution of signaling proteins. We demonstrate the power of this approach by comparing whole mount and conventional cryosection labeling of Kirrel2. This axon-guidance adhesion molecule is known to localize in a subset of sensory neurons and their axons in an activity-dependent manner. Whole mount cilia labeling revealed an additional and novel picture of the localization of this protein.

Cilia of olfactory sensory neurons contain proteins of the signal transduction cascade, but a detailed spatial analysis of their distribution is difficult in cryosections. We describe here an optimized approach for whole mount labeling and en face visualization of ciliary proteins.

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single ...

An Explant System for Time-Lapse Imaging Studies of Olfactory Circuit Assembly in Drosophila

~Neurons are precisely interconnected to form circuits essential for the proper function of the brain. The Drosophila olfactory system provides an excellent model to investigate this process since 50 types of olfactory receptor neurons (ORNs) from the antennae and maxillary palps project their axons to 50 identifiable glomeruli in the antennal lobe and form synaptic connections with dendrites from 50 types of second-order projection neurons (PNs). Previous studies mainly focused on identifying important molecules that regulate the precise targeting in the olfactory circuit using fixed tissues. Here, an antennae-brain explant system that recapitulates key developmental milestones of olfactory circuit assembly in culture is described. Through dissecting the external cuticle and cleaning opaque fat bodies covering the developing pupal brain, high quality images of single neurons from live brains can be collected using two-photon microscopy. This allows time-lapse imaging of single ORN axon targeting from live tissue. This approach will help reveal important cell biological contexts and functions of previously identified important genes and identify mechanisms underpinning the dynamic process of circuit assembly.

An Explant System for Time-Lapse Imaging Studies of Olfactory Circuit Assembly in Drosophila

This protocol describes the dissection procedure, culture condition, and live imaging of an antennae-brain explant system for the study of the olfactory circuit assembly.

~Neurons are precisely interconnected to form circuits essential for the proper function of the brain. The Drosophila olfactory system provides an ...

Developmental Biology

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. ...

Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system. They provide multiple experimental advantages, such as accessibility to numerous imaging approaches, electrophysiological techniques and behavioral assays. The Xenopus tadpole olfactory system is particularly well suited to investigate the function of synapses established during normal development or reformed after injury. Here, we describe methodologies to evaluate the processing of olfactory information in living Xenopus larvae. We outline a combination of in vivo measurements of presynaptic calcium responses in glomeruli of the olfactory bulb with olfactory-guided behavior assays. Methods can be combined with the transection of olfactory nerves to study the rewiring of synaptic connectivity. Experiments are presented using both wild-type and genetically modified animals expressing GFP reporters in central nervous system cells. Application of the approaches described to genetically modified tadpoles can be useful for unraveling the molecular bases that define vertebrate behavior.

Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system in vivo. We describe methodologies to evaluate the processing of olfactory information in living Xenopus larvae in normal rearing conditions or after injury.

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system. They provide multiple experimental advantages, such as ...

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization

Animals rely on chemical communication to convey and perceive relevant environmental information, ranging from assessment of food quality to detection of available mating partners or threats. In mice, this task is executed primarily by the olfactory system and its underlying subsystems, including the main and accessory olfactory systems. Both have peripheral organs populated by sensory neurons expressing G-protein coupled receptors able to bind chemical cues that reach the nasal cavity. Even though the molecular characteristics of these receptors is well understood, little is known about their cognate specific ligands. The method described here combines in situ hybridization detection of olfactory or vomeronasal receptors with immunodetection of phosphorylated ribosomal protein S6 (pS6) - a marker of neuronal activation. This protocol was devised to identify neurons activated after a single event of exposure to purified or complex chemical stimuli detected by the olfactory organs. Importantly, this technique allows the investigation of neurons triggered in biologically relevant contexts. Ideally, this method should be used to probe the molecular biology of the olfactory system and to study olfactory behaviors.

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization

This protocol combines in situ hybridization with immunofluorescence to identify olfactory and vomeronasal receptor genes expressed in olfactory sensory neurons after activation by chemical stimuli in the mouse.

Animals rely on chemical communication to convey and perceive relevant environmental information, ranging from assessment of food quality to detection of ...

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

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