Name: Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo (Video) | JoVE Journal
Uploaded: 2011-05-22T14:00:00.000+00:00
Duration: 11 min 34 s
Description: Learn lentivirus-mediated genetic manipulation for visualizing single olfactory sensory neuron axons and terminal arborizations in vivo. This method enables detailed study of individual neuron morphologies in the olfactory system.

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5 minutes

This study is supported by NIH DC052256 and DC006015, and NSF 0324769 to QG and T32-DC008072 to BS.

1. Preparation
This procedure is biosafety level 2, therefore all the following preparations are made in a biohazard hood where the microinjection procedure is performed.
<ol>
	<li>Prepare and preheat a 37°C waterbed: Fill a plastic storage bag with water – seal and set on an emptied heating block incubator. This will allow mice to recover following injection.</li>
	<li>Fill a biohazard waste container with 10% bleach to a suitable volume for immersion of all waste from this procedure.</li>
	<li>Prepare a small open dish of 70% ethanol and a second dish with sterilized ddH2O and place in the biological safety cabinet. </li>
	<li>Sterilize a 5μL capacity Hamilton syringe with a 33-gauge needle by spraying down the body and needle with 70% ethanol with the plunger pulled out all the way. Repeatedly aspirate ethanol from the dish and eject it all the way out at least 5 times.</li>
	<li>Rinse the syringe cartridge by aspirating and ejecting sterile water at least 5 times. Place syringe safely aside in the culture hood and allow it to dry.</li>
	<li>Remove viral stocks from -80°C freezer storage and place on ice to allow thawing. Microinjection requires 2μL of virus stock per mouse at a titre of 109 infectious units/mL. Lentiviruses can be obtained from commercial sources or produced in the lab according to the established protocol (Lois et al., 2002).</li>
	<li>Prepare an injection stage that provides an area raised above the hood surface - can use a 10cm culture dish covered with a sheet of Kimwipe.</li>
	<li>Arrange alcohol wipes near the injection station.</li>
</ol>
2. Microinjection of lentiviral particles into the olfactory epithelium
All animal procedures performed in this method are done according to IACUC approved guidelines. The Mus musculus strain C57BL/6J is the animal model used in all experiments demonstrated here.
<ol>
	<li>Anaesthetize mouse pups by hypothermia by taking a mouse pup (P0-P3) out of the cage and putting the pup on a cut-open glove on ice for 5 minutes.</li>
	<li>While waiting for the pup to be fully anesthetized, aspirate 2μL of viral stock into the sterilized Hamilton syringe.</li>
	<li>Place the mouse pup on top of the injection stage. Gently pull skin away from the top of the head at the ear toward the top of the neck. This should provide a taught skin surface over the nasal cavity and will ease injection.</li>
	<li>Insert needle through the top of the nasal cavity about 3mm below the skin surface to reach the olfactory epithelium. Microinject 0.5μL of virus solution per shot, choosing two sites per side, both left and right (four injections total per mouse = 2μL of virus needed). Injection sites should be staggered apart from one another. For targeting of proper injection sites, search the dorsal skull surface for the outline of the olfactory bulb. This line is barely visible at birth through the semi-translucent skin and skull. To make two injections per side, aim one injection just rostral to one bulb outline at a medial axis. Make the second insertion lateral to the bulb outline, yet make sure to angle the injection medially so that you may reach the olfactory epithelium that resides ventrally underneath the bulb. Avoid penetrating the olfactory bulb!</li>
	<li>Blot dry any residual virus solution or blood that may escape from the injection site or nostril openings using a Kimwipe. Immediately dispose of Kimwipes in the diluted liquid bleach biohazard container.</li>
	<li>Place the injected mouse onto the waterbed incubator.</li>
	<li>Repeat steps 1-4 for all mice to be injected, remembering you will need about 2μL of virus stock per mouse.</li>
	<li>Let mice recover on the waterbed incubator for 30 minutes before returning them to their original nesting cage.</li>
	<li>Dispose of microinjection stage, sanitizing wipes and any extra cleaning paper products in the liquid bleach biohazard disposal.</li>
	<li>Clean the Hamilton syringe by repeated aspiration and ejection of ethanol and then water as done in the preparation steps. Allow the syringe to dry.</li>
	<li>For maximum transduction efficiency repeat this procedure again in 4 hours. Infected OSNs may be visualized throughout the length of the cell in as few as three days.</li>
</ol>
3. Immunohistochemistry of floating olfactory bulb sections
<ol>
	<li>Sacrifice the injected mouse at the desired time point and perfusion fix the tissue with 4% paraformaldehyde. Dissect out the OB. Incubate in paraformaldehyde overnight and then 30% sucrose in PBS overnight. Embed in O.C.T. embedding medium on dry ice. </li>
	<li>Coronal OB sections are obtained at a 60μm in thickness using a cryostat. Sweep bulb sections into a 10cm culture dish with 5mL 1x PBS (pH 7.4). Sections should be kept floating in PBS to allow O.C.T. to dissolve. Transfer floating sections and PBS into a 50mL conical vial.</li>
	<li>Allow sections to settle to the bottom of the vial. Vacuum off PBS without sucking up any sections. Rinse tissue sections with 20mL PBS. Repeat settle and vacuuming steps. Resuspend tissue with 10mL PBS and transfer floating sections to a 15mL conical vial for immunostaining. Once tissue sections have settled, vacuum off PBS supernatant without disturbing tissue. Resuspend sections with 0.3% Triton X-100 in 1x PBS, pH 7.4 (PBS-Tx) allowing detergent to perforate cell membranes. Incubate for 10 minutes at room temperature. Vacuum off supernatant and repeat incubation with PBS-Tx twice.</li>
	<li>Incubate tissue sections in 10mL of blocking solution (5% horse serum in PBS-Tx) for two hours at room temperature.</li>
	<li>Rinse blocking solution at least twice with PBS-Tx for 15 minutes each at room temperature.</li>
	<li>Prepare 2mL of a primary antibody solution in PBS-Tx. See Reagents and Equipment section for antibodies used in this experiment.</li>
	<li>Replace second PBS-Tx blocking wash with primary antibody solution. Incubate on a slowly rocking platform (~30rpm) at 4°C for 24-48 hours to ensure thorough penetration into the 60μm floating sections.</li>
	<li>After incubation, the primary antibody solution can be retrieved carefully using a pipette and stored at 4°C for reuse up to two more times within two weeks without any significant loss of staining intensity.</li>
	<li>Wash sections three times in PBS-Tx for 20 minutes each at room temperature with gentle rotation (~30rpm).</li>
	<li>Prepare 2mL of secondary antibody dilution in PBS-Tx. Vacuum off the last wash and add secondary antibody solution to tissue sections. Wrap the outside of the staining vial in aluminum foil to block light, which can cause fluorophore photobleaching. Place wrapped vial on a slowly rocking platform and incubate at 4°C overnight.</li>
	<li>Discard secondary antibody solution by vacuuming away. Wash sections once with PBS-Tx for 20 minutes each at room temperature. Rinse two additional times with PBS (pH 7.4) for 20 minutes each at room temperature. Mount the stained tissue sections onto glass slides using Fluoromount G.</li>
	<li>Screen through stained sections for positively infected OSN axons and axon termini with a fluorescent microscope.</li>
	<li>To construct a three dimensional view of a labeled OSN axon and terminal arbor, make a series of optical sections through the 60μm depth of the target OB tissue slice (along the z axis) using confocal microscopy.</li>
	<li>This stack of Z-section images can subsequently be projected together to reconstruct a 360° rotating perspective of a single labeled OSN axon, including terminal arbor detail in its entirety. Any one single rotation angle can be captured and exported as an image file (i.e. JPG, TIFF) for 2D format presentation.</li>
</ol>
4. Representative Results
With the methods described here, we can transduce olfactory sensory neurons by lentivirus in vivo. We have thus far visualized infected OSNs with GFP and mCherry fluorescent reporters, as well with a myc-tagged fusion protein via immunocytochemistry. This technique provides an abundance of infected OSNs. Still, transduction occurs sporadically enough to provide individually labeled OSNs, thus allowing for analysis of single cell morphology. Axons and axon terminals are imaged within OB tissue sections amidst their native environment by confocal microscopy (Figure 1). The entire OSN axon terminal arbor can typically be captivated within a 40-50μm optical section. High magnification of infected OSN axon terminals allows detailed analysis of axonal arbor morphology at the glomerular layer.
<img src="/files/ftp_upload/2951/2951fig1.jpg" alt="Immunofluorescence microscopy of neural tissue showing OMP, VGluT2, GFP markers in various colors." data-altupdatedat="1787630408000"> 
Figure 1. Trajectory of single olfactory sensory neuron axon and its terminal arbor. A) A Coronal section of the olfactory bulb of a P7 mouse showing the olfactory nerve layer (ONL) immunostained for OMP (red) and the glomerular layer (GL) immunostained with both OMP and Vglut2 (grey). A single olfactory sensory neuron axon expressing GFP (green) travels within the ONL and penetrates into the glomerular structure. B) The axon terminal arbor morphology within the glomerulus clearly highlighted by GFP expression. Vglut2 co-staining in grey illuminates the total glomular area within which the GFP-positive axon elaborates a terminal abor. C) Lentiviral infected OSNs expressing GFP (green) in the OE. The cytostructure of the olfactory epithelium is shown with DAPI staining (blue). GFP expression allows visualization of OSN cell bodies and their cellular processes in the OE. D) An OSN axon and its terminal morphology with Rap1GAP2 expression knock-down by shRNA. Lentiviruses carrying a dual expression cassette with Rap1GAP2 shRNA under the mouse U6 promoter and GFP under a CMV promoter were applied to the OE. Infected olfactory axon terminals exhibited a more simplified terminal arbor when compared to that of the GFP only control. Bar = 20μm.

<ol><li>Chen, H., Kohno, K., Gong, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Conditional+ablation+of+mature+olfactory+sensory+neurons+mediated+by+diphtheria+toxin+receptor%5BTitle%5D)+AND+%22J+Neurocytol%22%5BJournal%5D)">Conditional ablation of mature olfactory sensory neurons mediated by diphtheria toxin receptor.</a> J Neurocytol. 34, 1-2 (2005).</li>
<li>Doi, K., Nibu, K., Ishida, H., Okado, H., Terashima, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adenovirus-mediated+gene+transfer+in+olfactory+epithelium+and+olfactory+bulb%3A+a+long-term+study%5BTitle%5D)+AND+%22Ann+Otol+Rhinol+Laryngol%22%5BJournal%5D)">Adenovirus-mediated gene transfer in olfactory epithelium and olfactory bulb: a long-term study.</a> Ann Otol Rhinol Laryngol. 114, 629-633 (2005).</li>
<li>Lois, C., Hong, E. J., Pease, S., Brown, E. J., Baltimore, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Germline+transmission+and+tissue-specific+expression+of+transgenes+delivered+by+lentiviral+vectors%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors.</a> Science. 295, 868-872 (2002).</li>
<li>Zhao, H., Otaki, J. M., Firestein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adenovirus-mediated+gene+transfer+in+olfactory+neurons+in+vivo%5BTitle%5D)+AND+%22J+Neurobiol%22%5BJournal%5D)">Adenovirus-mediated gene transfer in olfactory neurons in vivo.</a> J Neurobiol. 30, 521-530 (1996).</li>
</ol>

This protocol is bio-safety level 2 compliant. All appropriate workspace, personal protection, and viral waste containment measures have been established accordingly. All animal procedures were performed according to IACUC guidelines.

Microinjection of lentivirus results in permanent transfer of gene constructs into OSN genomic DNA. This approach allows us to perform short-term or long-term manipulations of candidate genes via overexpression or shRNA mediated knock-down. In addition, we can fluorescently label single OSNs in an existing transgenic mouse line to observe co-localization of odorant receptor populations. Microinjection is required for lentiviral transduction of OSNs. We have attempted flushing lentivirus suspension into the nasal cavity t...

<table><tbody><tr><td colspan="4">&lt;p class=&quot;jove_content&quot;&gt;&lt;em&gt;Microinjection:&lt;/em&gt; Newborn mice were microinjected using a 5&amp;#x03BC;L Hamilton syringe (Hamilton #7647-01) fitted with a 33 gauge needle (Hamilton #7762-06).&lt;/p&gt;&lt;p class=&quot;jove_content&quot;&gt;&lt;em&gt;Immunocytochemistry Reagents:&lt;/em&gt; Primary antibodies: rabbit polyclonal antibody GFP (Molecular Probes# A-6455), chicken polyclonal antibody against OMP (custom) (Chen et al., 2005), guinea pig polyclonal antibody against VGlut2 (Millipore# AB2251). Secondary antibodies: Cy2-conjugated donkey anti-rabbit (Jackson Immunolab# 711-225-152), Cy3-conjugated donkey-anti-chicken (Jackson Immunolab# 703-165-155) and Cy5-conjugated goat anti-guinea pig (Jackson Immunolab# 106-175-008). Sections were mounted on glass slides with Fluoromount G (Southern Biotech# 0010-01) with 50ng/mL DAPI chromatin stain solution.&lt;/p&gt;&lt;p class=&quot;jove_content&quot;&gt;&lt;em&gt;Confocal Imaging:&lt;/em&gt; Z-stack images were taken using an Olympus Flouview FV1000 confocal microscope and images collected and processed into 3D projections using Olympus FV10-ASW 2.01 confocal acquisition and analysis software.&lt;/p&gt;</td></tr></tbody></table>

lentivirus-mediated genetic manipulation and visualization of olfactory sensory neurons in vivo

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.

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 ...

One of the challenges of studying development in neuroscience is capturing dendritic and axonal morphologies at a single cell level in vivo. In the olfactory system, olfactory sensory neurons extend their axons from the olfactory epithelium in the nose to the olfactory bulb in the brain. As multiple olfactory sensory neurons project toward the olfactory bulb, they fasci into bundles.This complicates the study of individual axon trajectories and their terminal morphologies. We have developed a method to visualize single olfactory sensory neurons in vivo by micro injecting A GFP construct. Bearing lentivirus.Successful transduction with lentivirus illuminates the entire olfactory sensory axon length, including the terminal arbor. In addition to visualizing single olfactory sensory neurons, this method allows us to manipulate gene expression of a particular gene of interest to study its function in an in vivo context. This lentivirus procedure is biosafety level two and needs to be performed in a laminar flow hood.To maintain a sterile environment, spray everything inserted into the hood with 70%ethanol in the hood, prepare and preheat a 37 degree waterbed at least 20 minutes. Before injection. Seal a plastic storage bag half full with water and set it on an empty heating block incubator.This will allow mice to recover following injection. Fill a biohazard waste container with 10%bleach to decontaminate all virus exposed material. From this procedure, prepare a small open dish of 70%ethanol and a second dish with sterile distilled water.In a laminar flow hood, we'll be using a five microliter capacity Hamilton syringe with a 33 gauge needle. This particular model allows you to adjust the plunger resistance by turning a tension dial located at the top of the plunger chamber. This feature provides microinjection volume control to sterilize the needle and syringe chamber.Repeatedly aspirate and eject ethanol from the dish several times. Rinse the syringe cartridge by aspirating and ejecting sterile water several times. Place syringe safely aside in the flow hood.Arrange alcohol wipes near the injection station. Prepare an injection stage that provides a raised surface that will ease positioning of the mouse. One can use an inverted 10 centimeter culture dish covered with Kim wipes.Mouse pups can be anesthetized by hypothermia up until weaning age. Cut out a finger of a rubber glove and place the mouse pup in the finger cutting. Then place on ice for five minutes.The glove will prevent skin burns that would occur from direct skin contact with the ice test for thorough anesthesia by pinching the pup's hind and four feet to make sure the animal does not respond. We are now ready for microinjection of lentivirus. We'll be demonstrating with a neonatal pup.If an older age of mouse is used, one should substitute the 33 gauge needle for a thicker gauge needle in order to penetrate the skull's surface. Sterilize the mouse's nose surface by wiping it with an alcohol pad. Remove lentivirus stocks from freezer storage and thaw on ice.We have produced lentivirus in the lab ourselves using established methods. In developing this method, we have found that a minimal viral titer of 10 to the ninth infectious units per mil is required for a good transduction rate. Aspirate two microliters of lentivirus stock solution to the sterilized Hamilton syringe.Place the mouse pup on top of the injection stage. Grasp the skin on top of the head at the ears, and gently pull back. We have outfitted our flow hood with a dissecting scope.In order to accurately direct micro injections, search the dorsal skull surface at the forebrain for an outline of the olfactory bulb. Our micro injection targets closely surround the olfactory bulb, but should not include the bulb itself. We give four injections into the olfactory epithelium of each pup.These positions represented with blue dots require the needle to be inserted at an angle directed medially. The additional two injections represented with green dots are staggered, more medially and anterior to the olfactory bulb. These injections can be angled coddly.The needle angles suggestions are made in consideration of the ventral positioning of the olfactory epithelium, which is mostly situated beneath the olfactory bulb. Perpendicular access is thus restricted. Insert the needle through the top of the nasal cavity about three millimeters below the skin surface.To reach the olfactory epithelium. A trained hand should feel the drop in resistance when the nasal cavity luminal space is reached. This area should be avoided.Micro inject 0.5 microliters of virus stock solution per position. Again, choosing two sites per side, both left and right, four injections total of 0.5 microliters. Each per mouse means two microliters of virus needed per mouse in a single injection session.Again, be careful not to inject the olfactory bulb blot dry any residual virus solution or blood that may escape from the injection site or nostril openings. Using an ethanol wipe, dispose of wipes in the diluted liquid bleached container, immediately placed the injected mouse onto the waterbed incubator for recovery. Decontaminate needle syringe by repeating sterilization procedure performed during preparation Injected mice can be returned to their cage 30 minutes after recovery on the waterbed incubator.This entire microinjection can be repeated again in four hours. If you'd like to increase the number of olfactory sensory neurons that will be infected, infected olfactory sensory neurons may be visualized throughout the length of the cell in as few as three days. The animals sacrificed and perfused for observation at the desired time point.Using standard methods In order to visualize transduced single cell olfactory sensory neuron trajectories and axon termini, we dissect and section the olfactory bulb coronal at a 60 micron thickness. The section will thus contain all cell layers of the olfactory bulb, including olfactory sensory neuron axon bundles, and axon terminals, which synapse in glomerular structures bound at the bulb surface in preparation for floating immunohistochemistry. Serial sections of the olfactory bulb are made and collectively swept into a 10 centimeter culture dish with five milliliters of phosphate buffered saline.Transfer the floating sections into a 50 milliliter conical vial. Once sections have settled to the bottom of the vial, vacuum off phosphate buffered saline. Take caution not to vacuum up.Your sections replenish with 10 milliliters of fresh buffer to wash away any residual fixative or embedding material. Immediately pipette this resuspension into a 15 milliliter conical vial, making sure to capture all floating sections, vacuum off rinse solution, and perforate the sections with a solution of 0.3%Triton X 100 in phosphate buffer saline place on a rocker platform for 10 minutes. At room temperature, you may then proceed through a standard immuno staining protocol in the same conical vial, repeating the vacuum and rocker steps as needed for this size vessel, retain a 10 milliliter volume for staining and rinsing steps.Here is a list of antibodies we use to cos stain positively transduced olfactory sensory neurons, and the general olfactory sensory neuron population. For thorough antibody penetration of the 60 micron bulb sections, it is recommended to incubate overnight at four degrees Celsius. Wash depth should also be no shorter than 20 minutes each.Gently spread each section on a glass slide with a paintbrush mount each slide with flora Mount G supplemented with chromatin stain solution. Make efforts to eliminate all bubbles from the mounting medium. Mount your Sections in rows to simplify the subsequent screening step.Scan these layers on your immuno stain bulb sections for the GFP positive axon fibers and termini. Using a fluorescent microscope positively infected nerve terminals can then be imaged by confocal microscopy. It is helpful to calibrate the focal plane in your area of interest using the DPI and olfactory marker protein channels.These results show a single olfactory sensor neuron axon fiber and axon terminal within the glomerulus, which is CO with olfactory marker protein in red and vlu two in gray infected olfactory sensor neuron cell bodies and their dendritic knob can also be visualized within the olfactory epithelium if this tissue was included in the dissection and immunostaining steps. Hi, my confocal projection provides us with a greatly detailed morphology of a single olfactory sensory neuron axonal extension path and terminal arbor. One can see the hooking of the axon fiber back towards its glomerular target, as well as the details of its branching pattern within the glomerular neuro pill.The lentiviral microinjection method allows visualization of single cell axon, morphologies of olfactory sensory neurons, and allows studies that manipulate gene expression, lentivirus transduction results in stable genomic integration of constructs. Lentivirus does not trans synaptically infect neighboring cells. Visualization can be achieved in as few as three days and has been confirmed to last at least three months and likely persists for longer periods.Olfactory sensory neurons are not readily infected by nasal infusion of lentivirus solution. Therefore, microinjection through the surface is required in this procedure. So that's our lentivirus Microinjection procedure.I hope you've found all this information useful towards achieving single cell morphologies of your own Happy hunting.

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Learn lentivirus-mediated genetic manipulation for visualizing single olfactory sensory neuron axons and terminal arborizations in vivo. This method enables detailed study of individual neuron morphologies in the olfactory system.

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

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