Name: Viral Tracing of Genetically Defined Neural Circuitry (Video) | JoVE Journal
Uploaded: 2012-10-17T12:00:00.000+00:00
Duration: 13 min 6 s
Description: Learn viral tracing of neural circuitry using TVA receptor specificity and trans-synaptic virus in the central nervous system. This high-throughput method enables efficient mapping of synaptically connected neurons.

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We would like to acknowledge Sean Whelan for assistance with rescuing recombinant VSV variants, and Didem Goz and Ryan Chrenek for technical assistance. This work was supported by HHMI (CLC), and #NS068012-01 (KTB).

<ol><li>Wickersham, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Monosynaptic+restriction+of+transsynaptic+tracing+from+single%2C+genetically+targeted+neurons%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Monosynaptic restriction of transsynaptic tracing from single, genetically targeted neurons.</a> Neuron. 53, 639-647 (2007).</li>
<li>Marshel, J. H., Mori, T., Nielsen, K. J., Callaway, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+single+neuronal+networks+for+gene+expression+and+cell+labeling+in+vivo%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Targeting single neuronal networks for gene expression and cell labeling in vivo.</a> Neuron. 67, 562-574 (2010).</li>
<li>Wall, N. R., Wickersham, I. R., Cetin, A., De La Parra, M., Callaway, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Monosynaptic+circuit+tracing+in+vivo+through+Cre-dependent+targeting+and+complementation+of+modified+rabies+virus%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">Monosynaptic circuit tracing in vivo through Cre-dependent targeting and complementation of modified rabies virus.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 21848-21853 (2010).</li>
<li>Wickersham, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Monosynaptic+restriction+of+transsynaptic+tracing+from+single%2C+genetically+targeted+neurons%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Monosynaptic restriction of transsynaptic tracing from single, genetically targeted neurons.</a> Neuron. 53, 639-647 (2007).</li>
<li>Yonehara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spatially+asymmetric+reorganization+of+inhibition+establishes+a+motion-sensitive+circuit%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Spatially asymmetric reorganization of inhibition establishes a motion-sensitive circuit.</a> Nature. 469, 407-410 (2011).</li>
<li>Stepien, A. E., Tripodi, M., Arber, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Monosynaptic+rabies+virus+reveals+premotor+network+organization+and+synaptic+specificity+of+cholinergic+partition+cells%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Monosynaptic rabies virus reveals premotor network organization and synaptic specificity of cholinergic partition cells.</a> Neuron. 68, 456-472 (2010).</li>
<li>Beier, K. T., Samson, M. E. S., Matsuda, T., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Conditional+expression+of+the+TVA+receptor+allows+clonal+analysis+of+descendents+from+Cre-expressing+progenitor+cells%5BTitle%5D)+AND+%22Dev.+Biol%22%5BJournal%5D)">Conditional expression of the TVA receptor allows clonal analysis of descendents from Cre-expressing progenitor cells.</a> Dev. Biol. 353, 309-320 (2011).</li>
<li>Seidler, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Cre-loxP-based+mouse+model+for+conditional+somatic+gene+expression+and+knockdown+in+vivo+by+using+avian+retroviral+vectors%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">A Cre-loxP-based mouse model for conditional somatic gene expression and knockdown in vivo by using avian retroviral vectors.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 10137-10142 (2008).</li>
<li>Beier, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anterograde+or+retrograde+transsynaptic+labeling+of+CNS+neurons+with+vesicular+stomatitis+virus+vectors%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">Anterograde or retrograde transsynaptic labeling of CNS neurons with vesicular stomatitis virus vectors.</a> Proc. Natl. Acad. Sci. U.S.A. 108, 15414-15419 (2011).</li>
<li>Whelan, S. P., Ball, L. A., Barr, J. N., Wertz, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+recovery+of+infectious+vesicular+stomatitis+virus+entirely+from+cDNA+clones%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">Efficient recovery of infectious vesicular stomatitis virus entirely from cDNA clones.</a> Proc. Natl. Acad. Sci. U.S.A. 92, 8388-8392 (1995).</li>
<li>Fuerst, T. R., Niles, E. G., Studier, F. W., Moss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Eukaryotic+Transient-Expression+System+Based+on+Recombinant+Vaccinia+Virus+That+Synthesizes+Bacteriophage+T7+RNA+Polymerase%5BTitle%5D)+AND+%22PNAS%22%5BJournal%5D)">Eukaryotic Transient-Expression System Based on Recombinant Vaccinia Virus That Synthesizes Bacteriophage T7 RNA Polymerase.</a> PNAS. 83, 8122-8126 (1986).</li>
<li>Young, J. A., Bates, P., Varmus, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+a+chicken+gene+that+confers+susceptibility+to+infection+by+subgroup+A+avian+leukosis+and+sarcoma+viruses%5BTitle%5D)+AND+%22J.+Virol%22%5BJournal%5D)">Isolation of a chicken gene that confers susceptibility to infection by subgroup A avian leukosis and sarcoma viruses.</a> J. Virol. 67, 1811-1816 (1993).</li>
<li>Madisen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+robust+and+high-throughput+Cre+reporting+and+characterization+system+for+the+whole+mouse+brain%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">A robust and high-throughput Cre reporting and characterization system for the whole mouse brain.</a> Nat Neurosci. 13, 133-140 (2010).</li>
<li>Franklin, K., Paxinos, G. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aFranklin%2C+K+%22The+Mouse+Brain+in+Stereotaxic+Coordinates%22">The Mouse Brain in Stereotaxic Coordinates</a>. , Academic Press. San Diego, CA. (1997).</li>
<li>van den Pol, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Viral+strategies+for+studying+the+brain%2C+including+a+replication-restricted+self-amplifying+delta-G+vesicular+stomatis+virus+that+rapidly+expresses+transgenes+in+brain+and+can+generate+a+multicolor+golgi-like+expression%5BTitle%5D)+AND+%22J.+Comp.+Neurol%22%5BJournal%5D)">Viral strategies for studying the brain, including a replication-restricted self-amplifying delta-G vesicular stomatis virus that rapidly expresses transgenes in brain and can generate a multicolor golgi-like expression.</a> J. Comp. Neurol. 516, 456-481 (2009).</li>
</ol>

No conflicts of interest declared.

Using viruses to study neural circuits is a relatively high throughput method of analyzing connected neurons. However, generating both VSV and RABV virions is not trivial. Although the protocol listed above for rescuing virus from cDNA is provided, it is still a low-probability event. The levels of each of the N, P, and L plasmids need to be finely adjusted, and many trials and replicates need to be done to ensure viral rescue. The formation of the ribonucleotide particle incorporating a full VSV genome RNA is a low-prob...

<table><tbody><tr><td colspan="4">&lt;strong&gt;Tissue Culture&lt;/strong&gt;</td></tr><tr><td>Baby Hamster Kidney (BSRT7) cells</td><td>available upon request</td><td></td><td></td></tr><tr><td>vaccinia (vTF7-3)</td><td>available upon request</td><td></td><td></td></tr><tr><td>pN, pP, pl plasmids</td><td>available upon request</td><td></td><td></td></tr><tr><td>Calcium Chloride</td><td>Sigma</td><td>C1016</td><td></td></tr><tr><td>Magnesium Chloride</td><td>Sigma</td><td>M8266</td><td></td></tr><tr><td>HEK 293T cells</td><td>Open Biosystems</td><td>HCL4517</td><td></td></tr><tr><td>60 mm TC-Treated Culture Dish</td><td>Corning</td><td>430166</td><td></td></tr><tr><td>75 cm&lt;sup&gt;2&lt;/sup&gt; Rectangular Canted Neck Cell Culture Flask with Vent Cap</td><td>Corning</td><td>430641</td><td></td></tr><tr><td>Media : DMEM (Dulbecco&#039;s Modified Eagle Medium)</td><td>Invitrogen</td><td>12491-015</td><td></td></tr><tr><td>1 M HEPES pH 7.4</td><td>Gibo</td><td>15630-080</td><td></td></tr><tr><td>FBS: Fetal Bovine Serum</td><td>Gibco</td><td>10437-028</td><td></td></tr><tr><td>PKS</td><td>Invitrogen</td><td>15140-163</td><td></td></tr><tr><td>Lipofectamine 2,000 Transfection Reagent</td><td>Invitrogen</td><td>11668-019</td><td></td></tr><tr><td>Syringe: 5 ml Luer-Lock syringe</td><td>Sigma</td><td>Z248010-1PAK</td><td></td></tr><tr><td>Syringe Filters</td><td>Nalgene</td><td>190-2520</td><td></td></tr><tr><td>PEI: High Potency Linear PEI</td><td>Polysciences</td><td>23966</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Viral Centrifugation&lt;/strong&gt;</td></tr><tr><td>Corning 150 ml Tube Top Vacuum Filter System, 0.45 &amp;mu;m Pore</td><td>Corning</td><td>430314</td><td></td></tr><tr><td>Thinwall, Ultra-Clear, 38.5 ml, 25 x 89 mm ultracentrifuge tubes</td><td>Beckman-Coulter</td><td>344058</td><td></td></tr><tr><td>Ultracentrifuge</td><td>Beckman-Coulter</td><td>optima XL-80K</td><td></td></tr><tr><td>SW28 Ultracentrifuge rotor</td><td>Beckman-Coulter</td><td>342207</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Mouse Injection&lt;/strong&gt;</td></tr><tr><td>Capillary micropipets</td><td>Drummond</td><td>5-000-2005</td><td></td></tr><tr><td>Stereotax</td><td>Narishige</td><td>SR-5M</td><td></td></tr><tr><td>Micromanipulator</td><td>Narishige</td><td>SM-15</td><td></td></tr><tr><td>Ump injector</td><td>World Precision Instruments</td><td>Sys-Micro4</td><td></td></tr><tr><td>Four channel microcontroller</td><td>World Precision Instruments</td><td>UMP3</td><td></td></tr><tr><td>M.TXB Bench Motor with C.EMX-1 Dial Control, 115 Volt</td><td>Foredom</td><td>M.TXB-EM</td><td></td></tr><tr><td>H.10 Handpiece, Quick Change</td><td>Foredom</td><td>H.10</td><td></td></tr><tr><td>Step Drill, 0.5 mm</td><td>Foredom</td><td>A-58005P</td><td></td></tr><tr><td>Micr&amp;#8211;lectrode holder</td><td>World Precision Instruments</td><td>MEH2S</td><td></td></tr><tr><td>Ketamine</td><td>Henry 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viral tracing of genetically defined neural circuitry

Viral Tracing of Genetically Defined Neural Circuitry

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus (RABV), and more recently vesicular stomatitis virus (VSV), for studying circuitry, is becoming increasingly popular. These higher throughput methods use viruses that transmit between neurons in either the anterograde or retrograde direction.
Recently, a modified RABV for monosynaptic retrograde tracing was developed. (Figure 1A). In this method, the glycoprotein (G) gene is deleted from the viral genome, and resupplied only in targeted neurons. Infection specificity is achieved by substituting a chimeric G, composed of the extracellular domain of the ASLV-A glycoprotein and the cytoplasmic domain of the RABV-G (A/RG), for the normal RABV-G1. This chimeric G specifically infects cells expressing the TVA receptor1. The gene encoding TVA can been delivered by various methods2-8. Following RABV-G infection of a TVA-expressing neuron, the RABV can transmit to other, synaptically connected neurons in a retrograde direction by nature of its own G which was co-delivered with the TVA receptor. This technique labels a relatively large number of inputs (5-10%)2 onto a defined cell type, providing a sampling of all of the inputs onto a defined starter cell type.
We recently modified this technique to use VSV as a transsynaptic tracer9. VSV has several advantages, including the rapidity of gene expression. Here we detail a new viral tracing system using VSV useful for probing microcircuitry with increased resolution. While the original published strategies by Wickersham et al.4 and Beier et al.9 permit labeling of any neurons that project onto initially-infected TVA-expressing-cells, here VSV was engineered to transmit only to TVA-expressing cells (Figure 1B). The virus is first pseudotyped with RABV-G to permit infection of neurons downstream of TVA-expressing neurons. After infecting this first population of cells, the virus released can only infect TVA-expressing cells. Because the transsynaptic viral spread is limited to TVA-expressing cells, presence of absence of connectivity from defined cell types can be explored with high resolution. An experimental flow chart of these experiments is shown in Figure 2. Here we show a model circuit, that of direction-selectivity in the mouse retina. We examine the connectivity of starburst amacrine cells (SACs) to retinal ganglion cells (RGCs).

A method of tracing synaptically connected neurons is described. We use TVA specificity of an upstream cell to probe whether a cell population of interest receives synaptic input from genetically defined cell types.

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus ...

The overall goal of the following experiment is to develop a new technique for analyzing microcircuitry in the central nervous system. This is achieved by first defining a population of neurons for infection by expression of the TVA receptor. As a second step, the trans synaptic virus is injected into the CNS, which infects the primary neurons of choice.Next, the virus will only spread trans synaptically to connected neurons that express the TVA receptor. In order to examine if these neurons are connected, results are obtained to show that this technique can be used to analyze microcircuitry based on the validation of labeling known connected microcircuitry. The main advantage of this technique over existing methods like electrophysiology and microscopy, is that it is a high throughput method for analyzing neuronal circuitry.We first had the idea for this method when we read the first study of mono synaptically restricted viral tracing by wickersham and colleagues. We felt that viruses could be further engineered to study connected neurons in novel ways. Please consult the written protocol for details on recovery of VSV from CD NA using the VACCINIA T seven system to generate cells for passaging.VSV first aspirate the media from four 80%cofluent 10 centimeter plates of TV eight 800 cells plated the previous day and replaced with five milliliters of unsu. Supplemented DMEM transfect each plate with five micrograms of the plasmid, encoding the glycoprotein of choice according to standard procedures. After about six hours post transfection replace media with DMEM supplemented with 10%FBS incubate the plates overnight in a tissue culture incubator set to 34 degrees Celsius with 5%carbon dioxide to allow glycoprotein expression and surface accumulation.Following the incubation, aspirate the media and replace with five milliliters of DMEM plus 10%FBS then infect each plate with VSV containing the MA rabies G chimeric glycoprotein at a multiplicity of infection of 0.01 to 0.1 and incubate at 34 degrees Celsius overnight 24 hours later. Check the infection levels of each plate by fluorescence microscopy. If more than 50%of cells were infected, collect the media.Replace with five milliliters of fresh media and then freeze the collected media at minus 80 degrees Celsius. Repeat the collection procedure 24 hours thereafter for three days for a total of four days of collection. Freezing each aliquot of collected media after the final aliquot has fully frozen at minus 80 degrees Celsius, though all of the collected media aliquots at 37 degrees Celsius at this point, there should be 80 milliliters of collected media past the collected media through 0.45 micron filters on ice.Then aliquot 36 milliliters of virus into a Beckman centrifuge tube, and concentrate the supernatant by ultracentrifugation at 80, 000 times G for 90 minutes at four degrees Celsius. Make sure that the rotor is properly balanced following the centrifugation to count into a peaker containing 10%bleach and use an aspirator to remove liquid from the side of the tube. Then shake the tubes containing the virus at 120 RPM for one hour at four degrees Celsius and resuspend the viral pellet in the smallest volume possible, which is usually around 30 microliters.Once the virus is resuspended tighter the virus by infecting a 24 well plate of 2 9 3 T cells with a one to 1 million tenfold dilution series and titering about two days post-infection. According to standard procedures, virally infected cells can be detected by fluorescence. It's important to use the correct moline for these experiments.The goal is to have the TVA receptor expressed in the cell types into which one wants to map. This can be achieved by expression of the TVA receptor by viral means or through a transgenic allele. This experiment uses six to 10 week old triple transgenic mice.Mice expressing a conditional TVA allele will crossed a mice expressing a chat, Cree allele. Then these mice were crossed with a line that express a fluorescent TD tomato allele. The final transgenic mouse expresses TVA and a conditional TD tomato allele in all cells expressing Cree, including the starburst omicron cells of interest.And this expression can be visualized using fluorescence microscopy. Check the stereotaxic coordinates for the region of interest in a mouse brain atlas and prepare the stereotaxic apparatus by turning on the micro injector controller and moving the plunger and electrode holder into place. Then backfill the needle with mineral oil to provide an interface with the virus and place the needle on the plunger ensuring that there are no air bubbles in the needle.Haw the virus and place it on ice. Set the micro injector to withdraw and move the tube virus underneath the capillary on the micro injector. Lower the tip of the capillary into the virus and withdraw the necessary amount of virus for the experiment.After a preoperative dose of buprenorphine is given, the animal is appropriately anesthetized according to institutional guidelines. Perform a toe pinch and ensure that the reflex response is absent. Before beginning the procedure.Place the anesthetized mouse on a heating pad propped on a movable stand for positioning the mouse. Apply eye ointment to prevent drying of the cornea while the animal is anesthetized. Now, place the mouse in the stereotaxic apparatus by gently inserting the ear barss into the air canal.Ensure that the head is parallel to the base of the stereo attacks and does not move laterally. Next, adjust the bite bar to help stabilize the head. Remove the fur in the area of the incision.Then wash this area three times with ethanol, followed by three times with iodine. After making a sagittal incision in the skin with a pair of scissors to reveal the skull, push back the skin with homemade surgical hooks. Then learn up the micro capillary with bgma and use the dials to move the micro injector to the coordinates for the lateral gent nucleus.Once the LGN has been located, drill a small hole in the skull over the site of injection. Position the capillary over the hole and slowly lower it to a position just dorsal of the brain surface. This is the zero position of the dorsal ventral coordinate.The micro capillary should be sharp enough to puncture the mouse dura. So slowly lower it to a depth of 2.75 millimeters and inject the required amount of virus at a speed of around 100 liters per minute. Allow the needle to remain in the brain for about seven minutes to permit diffusion of the virus from the injection site and prevent the virus from moving up the needle.Tracted then very slowly over a period of two minutes, raise the needle from the injection site and outta the brain. Finally suture the surgical site and monitor the animals until they have recovered from anesthesia. Administer buprenorphine every 12 hours for two days.Generally, VSV trans synaptic spread can be observed around 24 hours post-injection. However, longer waiting times will result in more spread. After euthanizing the mouse using carbon dioxide inhalation, dissect out the eye into 4%formaldehyde and incubate for one hour, ensuring that the eye remains submerged.Then transfer the eye to a Petri dish containing PBS. Use a pair of number 55 forceps to quickly remove the cornea and loosen and dissect the retina away from other tissues. The whole mount analysis.Cut the retina on a glass slide so that it lays flat. Place silicon spaces around the edges of the glass slide cover with prolonged gold mounting media and then cover slip using a zero thickness glass cover slip for analysis of retina sections. Place the retina in 30%sucrose in PBS until the retina sub merges.Then place the retina in a mixture consisting of a one-to-one ratio of OCT to 30%sucrose solution for three hours. Finally, flash, freeze the retina and store at minus 80 degrees Celsius until ready to section. This image shows a time course experiment demonstrating infectivity of TVA 800 cells and 2 9 3 T cells by vsv A slash rg.The upper panels show TVA 800 cells at six hours one day and two days post infection. The lower panels show 2 9 3 T cells at the same time points. The virus spreads between TVA expressing cells in a matter of hours, whereas the spread on 2 9 3 T cells, which do not express the TVA receptor, though it does occur, happens much slower.The scale bar represents 100 microns. This image shows a representative infection of retinal ganglion cells of non TVA expressing mice. Animals were injected into the LGN with the vsv A slash RG virus.Pseudo typed with R-A-B-V-G and tissue was harvested two days post-injection. A sparse infection is seen here with the retinal ganglion cells indicated by yellow arrowhead. This image shows a denser infection.This can be achieved with a higher TI of virus and a good quality injection as seen here. Different types of retinal ganglion cells can be identified based on morphology including small arbor ganglion cells, type one melanopsin ganglion cells on direction selective ganglion cells or DS GCs and on off DS GCs. Here the scale bar represents 50 microns as seen in the following images from A TVA expressing mouse.VSVA slash RG transmission from on-off DS GCs to Starburst omics follows the expected pattern. The on-off DS GCs indicated with white arrowhead, with green fill transmit virus to multiple starburst omics indicated by yellow arrowhead. As seen here all green cells that are not DS GCs co label with the Cree reporter indicating that they are TVA expressing Starburst omics.The DS GCs indicated by white arrow heads with green fill and starburst omics indicated by yellow arrow heads stratifying the appropriate chat layers of the retinal inner plexiform layer or IPL, the cells to which the virus transmits include both on and off. Starburst omicron. The numbers in this figure indicate the layers of the IPL Once mastered, the entire experiment can be done in one or two days if it's performed properly.The speed of VSV is one of its major advantages following this procedure. Other methods like channel red, opsin based circuit mapping can be performed in order to validate the viral circuitry tracing and to analyze the properties of these synaptic connections. After watching this video, you should have a good understanding of most of the different steps involved in viral circuit tracing from growing virus to in vivo injection, to analyzing fluorescently labeled neurons.

viral-tracing-of-genetically-defined-neural-circuitry

Research

JoVE Journal

Neuroscience

We provide a practical guide for delivering tracers in vivo and use the spinocerebellar pathway as a model system to demonstrate essential steps for successful neuronal circuit analysis in mice. We describe in detail our versatile tracing protocol that exploits wheat germ agglutinin (WGA) conjugated to Alexa fluorophores.

Revealing Neural Circuit Topography in Multi-Color

Transsynaptic tracing has become a powerful tool for analyzing central efferents regulating peripheral targets through multi-synaptic circuits. Here we present a protocol that exploits the transsynaptic pseudorabies virus to identify and localize a functional brain circuit, followed by classical tract tracing techniques to validate specific connections in the circuit between identified groups of neurons.

Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling

Capitalizing on a binary genetic strategy we provide a detailed protocol for neural circuit tracing in mice that express complementary transsynaptic tracers after Cre-mediated recombination. Because cell-specific tracer production is genetically encoded, our experimental approach is suitable to study the formation and maturation of neural circuitry during murine embryonic brain development at a single cell resolution.

Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain

Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells and/or their adjacent ependymal cells in the adult ventricular-subventricular neurogenic niche which allows the separate analysis of cell autonomous and non-autonomous, niche-dependent effects on neural stem cells.

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

We describe a technique to label neurons and their processes via anterograde or retrograde tracer injections into brain nuclei using an in vitro preparation. We modified an existing method of in vitro tracer electroporation by taking advantage of fluorescently labeled mouse mutants and basic optical equipment in order to increase labeling accuracy.

Laser-guided Neuronal Tracing In Brain Explants

This protocol describes large-scale reconstructions of selective neuronal populations, labeled following retrograde infection with a modified rabies virus expressing fluorescent markers, and independent, unbiased cluster analyses that enable comprehensive characterization of morphological metrics among distinct neuronal subclasses.

Large-scale Reconstructions and Independent, Unbiased Clustering Based on Morphological Metrics to Classify Neurons in Selective Populations

Based on in vitro lentiviral engineering of neuronal precursors, their co-transplantation into wild-type brains and paired morphometric evaluation of "test" and "control" derivatives, this method allows accurate modeling of in vivo gene control of neocortical neuron morphology in a simple and affordable way.

Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies

This protocol aims at generating directly reprogrammed interneurons in vivo, using an AAV-based viral system in the brain and a FLEX synapsin-driven GFP reporter, which allows for cell identification and further analysis in vivo.

In Vivo Direct Reprogramming of Resident Glial Cells into Interneurons by Intracerebral Injection of Viral Vectors

The current protocol demonstrates a simple method for tracing of ventral tegmental area (VTA) glutamate projections to the hippocampus. Photostimulation of VTA glutamate neurons was combined with CA1 recording to demonstrate how VTA glutamate terminals modulate CA1 putative pyramidal firing rate in vivo.

Combined In Vivo Anatomical and Functional Tracing of Ventral Tegmental Area Glutamate Terminals in the Hippocampus

Here we present a protocol describing viral transduction of discrete brain regions with optogenetic constructs to permit synapse-specific electrophysiological characterization in acute rodent brain slices.

Ex Vivo Optogenetic Interrogation of Long-Range Synaptic Transmission and Plasticity from Medial Prefrontal Cortex to Lateral Entorhinal Cortex

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. Many genes are involved in this process. This protocol introduces the technique for the time-lapse imaging of migrating neurons and glial progenitors.

Time-Lapse Imaging of Migrating Neurons and Glial Progenitors in Embryonic Mouse Brain Slices

Learn viral tracing of neural circuitry using TVA receptor specificity and trans-synaptic virus in the central nervous system. This high-throughput method enables efficient mapping of synaptically connected neurons.

Viral Tracing of Genetically Defined Neural Circuitry

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