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&deg;C waterbed: Fill a plastic storage bag with water &#x2013; 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 5&mu;L 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&deg;C freezer storage and place on ice to allow thawing. Microinjection requires 2&mu;L 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 2&mu;L 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 0.5&mu;L of virus solution per shot, choosing two sites per side, both left and right (four injections total per mouse = 2&mu;L 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 2&mu;L 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 60&mu;m 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&deg;C for 24-48 hours to ensure thorough penetration into the 60&mu;m floating sections.</li>
	<li>After incubation, the primary antibody solution can be retrieved carefully using a pipette and stored at 4&deg;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&deg;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 60&mu;m 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&deg; 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 40-50&mu;m 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" /> 
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 = 20&mu;m.

<ol>
	<li>Chen, H., Kohno, K., Gong, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+ablation+of+mature+olfactory+sensory+neurons+mediated+by+diphtheria+toxin+receptor.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-mediated+gene+transfer+in+olfactory+epithelium+and+olfactory+bulb:+a+long-term+study.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germline+transmission+and+tissue-specific+expression+of+transgenes+delivered+by+lentiviral+vectors.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-mediated+gene+transfer+in+olfactory+neurons+in+vivo.">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...

Microinjection: Newborn mice were microinjected using a 5&mu;L Hamilton syringe (Hamilton #7647-01) fitted with a 33 gauge needle (Hamilton #7762-06).
Immunocytochemistry Reagents: 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.
Confocal Imaging: 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.

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.

Watch this Scientific Journal Video about Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo at JoVE.com

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.

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 ...

lentivirus-mediated-genetic-manipulation-visualization-olfactory

Research

JoVE Journal

Neuroscience

15.6K visualizações.  University of California, Davis. Nós apresentamos uma técnica lentiviral para a manipulação genética e visualização de axônio único neurônio olfactory sensoriais e sua arborização terminal In vivo.

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

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 ...

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. ...

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 ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

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 ...

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 ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an excellent model system for such investigations due to its complex behaviors, powerful genetic techniques, and compact nervous system. Laboratory behavioral assays have long been used with Drosophila to simulate properties of the natural environment and study the neural mechanisms underlying the corresponding behaviors (e.g. phototaxis, chemotaxis, sensory learning and memory)1-3. With the recent availability of large collections of transgenic Drosophila lines that label specific neural subsets, behavioral assays have taken on a prominent role to link neurons with behaviors4-11. Versatile and reproducible paradigms, together with the underlying computational routines for data analysis, are indispensable for rapid tests of candidate fly lines with various genotypes. Particularly useful are setups that are flexible in the number of animals tested, duration of experiments and nature of presented stimuli. The assay of choice should also generate reproducible data that is easy to acquire and analyze. Here, we present a detailed description of a system and protocol for assaying behavioral responses of Drosophila flies in a large four-field arena. The setup is used here to assay responses of flies to a single olfactory stimulus; however, the same setup may be modified to test multiple olfactory, visual or optogenetic stimuli, or a combination of these. The olfactometer setup records the activity of fly populations responding to odors, and computational analytical methods are applied to quantify fly behaviors. The collected data are analyzed to get a quick read-out of an experimental run, which is essential for efficient data collection and the optimization of experimental conditions.

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

We describe here a behavioral setup and data analysis method for assaying olfactory responses of up to 100 vinegar flies (Drosophila melanogaster). This system may be used with single or multiple olfactory stimuli, and adaptable for optogenetic activation or silencing of neuronal subsets.