Name: viral-mediated labeling and transplantation of medial ganglionic eminence (mge) cells for in vivo studies
Uploaded: 2015-04-23T14:00:00.000+00:00
Duration: 9 min 21 s
Description: Watch this Scientific Journal Video about Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence MGE Cells for In Vivo Studies at JoVE.com

This work was supported by grants to JLRR from: Autism Speaks, Nina Ireland, Weston Havens Foundation, NIMH R01 MH081880, and NIMH R37 MH049428. PRW was supported by a fellowship from the National Science Council of Taiwan. SFS was supported by F32 (MH103003).

Ethics statement: The following procedures have been approved by our institution and animal protocol. Make sure to get approval for all procedures involving survival surgeries before beginning experiments and verify all protocols are up to date.
1. Lentivirus Preparation (Optional Step)
<ol>
	<li>Split three 10 cm plates of HEK293T cells for each lentivirus to be made, in the presence of 10 ml DMEM/10% FBS, and grow to ~60-70% confluence. Grow cells in an incubator at 37 &#176;C with 5% CO2.</li>
	<li>Transfect the following DNA plasmids (10 &#181;g total) into each plate using any transfection reagent: 6.4 &#181;g CAG-Flex-GFP lentiviral vector (DNA vector sequence provided in Table 1), 1.2 &#181;g pMD2.G (encodes VSV-g), 1.2 &#181;g pRSV-Rev, and 1.2 &#181;g pMDLg/pRRE. The three lentiviral packaging vectors are commercially available (Table 
	Note: This particular approach utilizes 3rd generation lentiviral vectors but 2nd generation vectors and associated plasmids will also work.</li>
	<li>Prepare a waste container containing a 10% bleach solution within a BSL2 certified hood to inactivate any solutions or devices that contacted or contain lentivirus. Next, completely remove the media 4-6 hr&#160;after transfection into the bleach solution, replace with the same volume of new media, and allow the cells to grow.</li>
	<li>After 4 days, collect media into 50 ml conical tubes and centrifuge at 1,000 x g for 15 min to pellet any cell debris. Next, filter the supernatant through a 0.45 &#181;m membrane. Any low protein binding filter, like PVDF, works well. Caution: place both solid and liquid viral waste into the bleach solution.</li>
	<li>Load 30 ml filtered supernatant into ultracentrifuge tubes and into an ultracentrifuge. Centrifuge at 100,000 x g for 2.5 hr at 4 &#176;C. Open the centrifuge tubes in a BSL2 hood and remove supernatant into 10% Bleach. Add ~100 &#181;l of PBS to the pellet, which yields titers of 1x10^7-8 infectious units/ml. Aliquot the following day and store at -80 &#186;C. Lentivirus aliquots are stable at -80 &#176;C for at least six months.</li>
	<li>Important consideration: Many institutions now have viral cores. Acquire concentrated virus with a minimum titer of 1x107 infectious units/ml, for optimal results.</li>
</ol>
2. Donor Mice for MGE Dissection
<ol>
	<li>First, set up a timed mating between a Cre-expressing line of interest and either a wild type (WT) mouse or a mouse that will express a reporter after Cre-mediated recombination. 
	Note: Many Cre-driver lines are transgenics and are maintained and bred in the hemizygous state. Thus, only half of the embryos will contain the Cre transgene. DNA can be quickly prepared from the embryos for a short PCR to detect the Cre allele. The Cre+ embryos can then be harvested so that only the MGE tissue of the desired genotype is used. Alternatively, a Cre-dependent reporter may be used, to select the embryos for MGE dissection and transplantation.</li>
	<li>Calculate which day will correspond to E13.5. If animals were paired the evening before, the day of the plug is 0.5. Order timed-pregnant mice or a WT female with P1 pups in order to time having postnatal (P)1 mice on the day the donor tissue will be E13.5. Alternatively, set up these matings in house one week prior to pairing the donor animals. 
	Note: The former strategy of generating both E13.5 and P1 embryos/pups for the same day is often difficult to do reliably.</li>
</ol>
3. Preparation of Media, Tools and Equipment.
<ol>
	<li>Prepare the reagents and tools for MGE cell preparation (Table 2).
	<ol>
		<li>Prepare a clean surface and place clean forceps, scissors and either a microsurgical knife or fine forceps (for precise tissue dissection) on the surface.</li>
		<li>Prepare Hank&#39;s balanced salt solution (HBSS), for dissection, and Dulbeco&#39;s modified eagle&#39;s medium (DMEM) with 10% fetal bovine surem (FBS), for transduction.</li>
		<li>Preincubate 20-30 ml DMEM/10% FBS in a 37 &#176;C tissue culture incubator for about 1 hr. The lid should be loosened to allow for gas exchange and pH equilibration of the media. 
		Note: The media preincubation can be prepared just before starting the dissection.</li>
	</ol>
	</li>
</ol>
4. MGE Cell Preparation
<ol>
	<li>Sacrifice the pregnant mouse according to an approved animal protocol and remove the embryos into a 10 cm petri dish containing ice-cold HBSS.</li>
	<li>Remove the brain from each embryo using a dissecting scope (do one at a time, leaving the rest of the embryos in ice-cold HBSS) and then proceed to cut out the MGE tissue. 
	Note: Detailed below, and in Figure 1, are key steps showing how to remove the MGE. In addition, Figure 2 is a movie showing the entire dissection procedure.
	<ol>
		<li>Position the brain with ventral side facing up (Figures 1A, A&#39;). Note: it is also okay to have the dorsal aspect facing up. Cut the brain in half along the saggital plane to generate two pieces. An example hemisected brain is shown (Figures 1B, B&#39;), notice that dorsal (the overlying cortex) and ventral aspects of the brain cover and surround the ganglionic eminence (GE) tissue.</li>
		<li>Next, with a dominant hand, hold either very fine forceps or a stabknife (Table 2), while immobilizing the hemisphere with forceps held by a non-dominant hand. (The hemisphere&#39;s medial aspect should be facing up). Unfold the dorsal and ventral tissue with forceps and a stab knife to reveal the underlying GE tissue (Figure&#160;1C and diagrammed in Figure&#160;1C&#39;). 
		Note: Removing some media from the dissection petri dish can make it easier to stabilize the tissue while performing the MGE dissection.</li>
		<li>Make straight cuts with the stabknife or fine forceps to separate the CGE, LGE and septal tissues (see example cuts denoted by red dashed lines, Figures 1D and D&#39;). These cuts can be made in any order. 
		Note: Imagine the MGE as a &#39;box&#39; that is cut out of the surrounding tissue. Doing so helps reproducibly make similar cuts for each dissection, thus reducing variability in tissue dissections.</li>
		<li>Finally, turn the MGE on its side and trim off the bottom (what was the lateral aspect of the brain). This removes the mantle zone of the MGE (discard in Figures 1E and 1E&#39;) from the rest of the MGE. The remaining aspect of MGE contains primarily ventricular zone and subventricular zone portions of the MGE, which contains nearly all the MGE progenitor cells. Proceed with this tissue.</li>
	</ol>
	</li>
	<li>Important consideration: The addition of silicone gel to the bottom of a petri dish can serve as an excellent dissection surface as it protects the delicate tips of forceps and provides a soft surface for pinning tissue with forceps.</li>
</ol>
4.3&#160;Additional strategies for MGE Dissection.
<ol>
	<li>Cut the skin of the embryo using forceps like scissors to quickly remove the brain. As the embryo lays on its side, hold the tissue at the base of the head with the forceps used to anchor the tissue. First, cut anterior ventral to the brain and posterior ventral to the brain, and then connect the two cuts along the side of the embryo. Then grab the flap of skin and pull it over the top of the brain. The brain can then be quickly dissected away from the remaining tissue.</li>
	<li>Alternatively, anchor the posterior aspect of the whole brain behind the cortex. Meanwhile, grasp each hemisphere at the most dorsal caudal aspect of the cortex and tear the cortex away from the anchored tissue in an anterior direction. In this way, both cortices can be removed and the bilateral ganglionic eminences are revealed, still attached to the rest of the tissue preserving the medial-lateral anatomy.</li>
	<li>Switch to the stab-knife to make precise cuts around the MGE on each hemisphere. Use either a separate clean set of forceps to collect the MGE or a pipette with a large tip (i.e., P1000). 
	Note: Keep the collected tissue away from all other dissected material, as some media is unavoidably transferred when the MGE is collected.</li>
	<li>Collect the MGE and put the tissue into a 1.5 ml collection tube containing DMEM/10% FBS, (a volume of 500 &#181;l&#160;is sufficient to collect the MGEs). Repeat for the other hemisphere and place in the same collection tube. Keep samples on ice until all tissue is collected.</li>
</ol>
5. Lentiviral labeling and Transplantation
<ol>
	<li>Move the tubes of MGE tissue into a BSL2 certified hood and remove the media. 
	Note: The MGE tissue is big enough to be visible to the eye and will settle to the bottom of the tube allowing for the cold media to be easily and gently removed.</li>
	<li>Add ~500 ul&#160;of DMEM/10% FBS media, that was preincubated in a 37 &#176;C tissue culture incubator. Next, add polybrene to facilitate transduction to each tube at a final concentration of 8 &#181;g/ml. Triturate the MGE tissue to create a single cell suspension, using a P1000 pipette tip. Finally, add ~15-20 &#181;l of concentrated lentivirus to each tube.</li>
	<li>Important consideration: A trituration of 10-12 times is sufficient to break up MGE tissue from a single embryo, but more triturations may be required if multiple MGEs are pooled together. Try out different trituration conditions to determine what works best.</li>
	<li>Securely close each tube, invert to mix, than place all the tubes in a 37 &#176;C incubator. Incubate the cells in lentivirus for at least 30 min and up to 1 hr. Longer times have resulted in decreased cell viability. Invert the tubes every 10 min until the time is up.</li>
	<li>After incubation with lentivirus, remove the tubes from the incubator and centrifuge at ~700 x g for 3 min to pellet the cells. In the BSL2 hood, remove the supernatant and discard into 10% bleach. Next, add 1 ml DMEM/10% FBS and triturate the pellet 2-3 times to wash, then centrifuge again at the same speed to pellet the cells. Repeat this wash-step 2-3 more times to remove excess virus. 
	Note : Perform centrifugation steps at 4 &#176;C, however, reduced viability has not been observed when short spins are performed at RT.</li>
	<li>After the final wash, remove as much media as possible and put each tube on ice. Due to residual media on the sides of the tube, ~2-3 &#181;l of media will end up covering the cell pellet by the time the transplantation procedure has begun.</li>
</ol>
6. Transplantation and Validation
<ol>
	<li>Acquire host P1 pups and prepare the procedure area by spraying down with 70% ethanol. To maintain sterility during the procedure it is recommended to perform these procedures in a dedicated clean procedure room. In addition, use either autoclaved surgical tools sterilize surfaces that the pups will be in contact with using 70% ethanol. An example of representative equipment needed to perform the transplantations and a representative procedure area is shown in Figure 3.</li>
</ol>
Note: While this procedure is an injection of small volumes, and not a surgical procedure that would require an incision, open wound or sutures, it is still recommended that a new micropipette is used for each mouse to be injected. The micropipettes are heat sterilized when pulled and beveled on a surface that was sprayed with 70% ethanol being stored in a sealed container.
<ol>
	<li>Generate micropipettes to have a diameter at the tip between 40-80 &#181;m. Following these dimensions will yield optimal results. Heat sterilize the micropipettes when pulled and beveled on a surface and spray with 70% ethanol before storing the micropipettes in a sealed container. 
	Note: Alternatively, if access to the devices needed to create these needles (Table 2) is limiting, use any other injection device. However, these may not be as well suited as the micropipettes due to different diameters.</li>
	<li>Draw up mineral oil into a 1 ml syringe, and with a 30&#160;1/2 G needle, fill the glass pipette completely with mineral oil. Next, mount the plunger onto the stereotaxic device, then attach the glass pipette to the stereotaxic device and load onto the plunger. Using the hydraulic drive, move the plunger about halfway into the glass pipette, removing mineral oil that spills out with a clean a clean paper towel.
	<ol>
		<li>Alternatively to using a syringe, submerge the back end of the pipette in mineral oil and filled by capillary action. To prevent air bubbles in the pipette, maintain positive pressure on the syringe until completely out of the glass pipette. 
		Note: Other devices can be used to successfully transplant MGE cells 14. Any device that can deliver a volume less than or near 100 nl works well for this procedure.</li>
	</ol>
	</li>
	<li>Next, use a sterile paper towel, or any absorbent material, with the corner twisted into a fine point to delicately remove excess media above the MGE cell pellet. This step concentrates the cell density so that smaller injection volumes can be achieved. Next, use a low volume (P2 is preferred) pipette to slowly draw up the cell pellet and triturate 1-2 times before moving the cell suspension onto a hydrophobic surface. The volume should be just under 1 &#181;l. Move the tip of the needle into the cell suspension and using the hydraulic drive, draw up the cell suspension into the pipette.</li>
	<li>&#160;Anaesthetize a P1 pup via hypothermia (wrap in a latex glove and put on ice for ~ 2-4 min). Check for effectiveness of anesthesia with a noxious stimulus. Pinch the skin between the toes with fine forceps: the pup should be unresponsive if pinched. Next, place the pup onto a mold that is under the injection device, then make the skin taut by pulling back the skin on the head and securing with standard lab tape. 
	Note: While hypothermia is very effective on neonatal mice and results in low mortality rate, procedures must be done quickly, as pups will begin to recover in under 10 min. In addition, 4 min on ice is an upper limit of what can be tolerated. Try to only induce hypothermia once if a longer time is required for injections. However, if a pup recovers early, first allow the pup to fully recover before inducing hypothermia again. Moreover, only induce hypothermia one more time to perform injections.&#160;&#160;Pups will recover within 5-10 min&#160;of hypothermia. This can be facilitated by placing the pups with litter and mom or by placing the pup on a warm surface to recover.</li>
	<li>Using a stereotaxic device, position the tip of the glass pipette on the surface on the pup&#39;s head, perpendicular to the surface of the head. When the pipette is in contact with the head, consider this as &#39;0&#39;. Next, push the pipette through the surface of the skin and skull into the cortex, then insert and retract the pipette ~ 2 times before stopping. For optimal targeting of cells into the neocortex, injections are performed when the micropipette is at a depth of 0.1 mm. However, this should be optimized by the user (see note below). 
	Note: While this depth reliably targets deeper neocortical layers with the devices described above, a few depths should be tested determined empirically. In addition, a range of depths at different rostro-caudal and medio-lateral position should be tested to determine what depth is optimal at each site. Also, the initial puncture should be rapid, as slow penetration into the neocortex diminishes the ability to cleanly penetrate the tissue. Try different speeds when first starting this procedure to find what works best. In addition, for testing coordinates, there is no real substitute for using labeled cells, as dyes have been unreliable to denote position.</li>
	<li>Once the needle is in position, rotate the hydraulic device to advance the plunger into the glass pipette, pushing a set volume of cell suspension into the cortex. Inject 50-70 nls per site. Repeat this procedure at 3-6 different sites at different rostral-caudal levels if the goal is to obtain a wide distribution of cells throughout the neocortex. 
	Note: Volumes of 100 nl or greater can cause lesions and lead to large clumps of injected cells that fail to efficiently migrate out of the injection site. Thus, smaller injection volumes (~70 nl or less) are optimal, over more sites if time permits.</li>
	<li>When injections are complete, remove the pup from the stage and mark it (toe clipping is a reliable way to identify the pups later). Once the pup has recovered and is able to move on its own (this occurs within minutes of removing them from the stage), put it back with the mom and litter. 
	Note: Since this is a minimally invasive procedure there are no post-surgical procedures once the pup is freely moving and warmed. However, pups should be checked not only after the procedure but also the following day to assure they have no signs of deteriorating health.</li>
	<li>Before starting the procedure on the next pup, spray down the area with 70% ethanol&#160;to maintain a sterile working area. Allow the injected pups to develop and then assess the tissue at the appropriate developmental stage(s).</li>
</ol>

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The authors declare that they have no competing financial interests.

The use of GABAergic cortical interneuron precursors from the embryonic ganglionic eminences (GEs) for cell based therapies is showing promise for many conditions 12&#8211;14. Precise molecular techniques are needed to track, and express genes of interest in specific interneuron subgroups. Here we provide a detailed protocol for labeling embryonic MGE cells with lentiviruses before transplantation and show how this technique can be used to express genes of interest in specific cortical in...

GABAergic cortical interneurons comprise ~20-30% of neurons in the mammalian neocortex, while the rest correspond to excitatory, glutamatergic principal neurons. Interneurons are highly diverse in electrophysiological properties, axon and dendrite morphology and synaptic targeting 1, and imbalances in excitatory/inhibitory tone are hypothesized to underlie some phenotypes of neurological/neuropsychiatric disorders including autism, schizophrenia and epilepsy 2. The overall goal of the protocol described herein is to provide a means to efficiently genetically modify GABAergic cortical interneuron progenitors before transplantation for in vivo analyses.
Cortical GABAergic interneurons are born in the medial and caudal ganglionic eminences (MGE and CGE, respectively) 3,4 as well as the preoptic area 5. Cortical interneuron progenitors undergo long-distance tangential migration followed by radial migration to reach their final targets. Upon arrival at their destinations, these cortical interneurons must correctly integrate into the existing neuronal network, and each unique interneuron subgroup will contribute to cortical circuitry in specific ways. Four main subgroups can be distinguished by molecular markers: MGE-derived somatostatin (SST)+ and parvalbumin (PV)+ subgroups, and CGE-derived vasoactive intestinal peptide (VIP)+ and Reelin+;SST- subgroups 6. Different cortical interneuron subgroups are born over different times during embryonic development in the MGE and CGE 7, 8. These and other cortical GABAergic interneuron markers have been used to generate specific Cre-driver lines for many of these subgroups 9-11.
The transplantation of MGE progenitors has emerged as a potential cell-based therapy to treat disorders that may be caused by imbalances in excitation/inhibition 12&#8211;24. These therapeutic benefits may be due in part to the unique ability of MGE progenitors (to disperse, differentiate and integrate into a host brain), or potentially because many peri-somatic inhibitory PV+ cells are derived from the MGE. MGE cells can also be quickly and efficiently transduced with lentiviruses before transplantation 15, allowing cells that are genetically modified in vitro to be studied in vivo. The rationale for developing this approach was to overcome roadblocks in studying GABAergic cortical interneuron development and maturation. In particular, MGE transplantation allowed researchers to study the development of mutant cells in vivo, when the mutant mouse would have otherwise died at an early time point. Moreover, by introducing genes of interest before transplantation, the effects of specific genes on a mutant phenotype could be assessed in an efficient manner.
Here, we provide a detailed protocol to transduce MGE cells with lentiviruses prior to transplantation. In addition, we show how this technique can be adapted to express a gene of interest in specific interneuron subgroups from a heterogeneous group of cortical interneuron precursors, using a combination of Cre-dependent expression lentiviruses and available Cre-driver mouse lines. Moreover, this protocol introduces techniques and a platform for researchers to genetically modify GABAergic cortical interneuron precursors for in vivo studies in a unique way. One advantage of this technique over other current approaches is that the transplanted MGE cells will disperse away from the injection site. Also, unlike focal viral injections, after MGE cells disperse their morphology is easier to assess. This approach can be used to study the effect of introducing genes of interest into wild type or mutant cells, introducing a cell type specific reporter to assess morphology, or potentially to study the effect of disease alleles in vivo.

<table><tbody><tr><td colspan="4">Reagents and equipment for MGE cell transplantation</td></tr><tr><td>1 ml syringe</td><td>REF 309602</td><td>BD, Becton Dickinson and company</td><td></td></tr><tr><td>30 1/2 G needle</td><td>305106</td><td>BD, Becton Dickinson and company</td><td></td></tr><tr><td>Precision bore to deliver 5 &amp;micro;l (comes with plunger)</td><td>5-000-1005</td><td>Drummond scientific company</td><td></td></tr><tr><td>Parafilm</td><td>PM-999</td><td>Polysciences, Inc.</td><td></td></tr><tr><td>Stereo microscope with boomstand **</td><td>MZ6</td><td>Leica</td><td></td></tr><tr><td>Digital just for mice stereotaxic instrument **</td><td>51725D</td><td>Stoelting company</td><td></td></tr><tr><td>Single-axis oil hydraulic fine micromanipulator **</td><td>MO-10</td><td>Narishige</td><td></td></tr><tr><td>Diamond coated rotary beveler</td><td></td><td>made in house</td><td></td></tr><tr><td>Needle pipette puller</td><td>730</td><td>Kopf instruments</td><td></td></tr><tr><td>Mineral oil</td><td></td><td>Any drug store or pharmacy</td><td></td></tr><tr><td>Any pipette and tips that can reliaby measure 1 &amp;micro;l of volume</td><td></td><td></td><td></td></tr><tr><td colspan="4">** These are the particular models we use, but many other setups should work</td></tr><tr><td colspan="4">Optional Reagents (for making Lentiviruses)</td></tr><tr><td>25 mm, 0.45 &amp;micro;m filter</td><td>09-719B</td><td>Fisher Scientific</td><td></td></tr><tr><td>Ultra-clear centrifuge tubes (25 x 89 mm)</td><td>344058</td><td>Beckman Coulter&amp;reg;</td><td></td></tr><tr><td>Lipofectamine &lt;sup&gt;2000 &lt;/sup&gt;(1.5 ml size)</td><td>11668019</td><td>Invitrogen</td><td></td></tr><tr><td colspan="4">Other commercially available supplies</td></tr><tr><td>pMDLg/pRRE plasmid encoding gag/pol</td><td>12251</td><td>Addgene</td><td></td></tr><tr><td>pRSV-Rev plasmid encoding Rev</td><td>12253</td><td>Addgene</td><td></td></tr><tr><td>pMD2.G plasmid encoding VSVG</td><td>12259</td><td>Addgene</td><td></td></tr><tr><td>pAAV-Flex-GFP</td><td>28304</td><td>Addgene</td><td></td></tr></tbody></table>

viral-mediated labeling and transplantation of medial ganglionic eminence (mge) cells for in vivo studies

GABAergic cortical interneurons, derived from the embryonic medial and caudal ganglionic eminences (MGE and CGE), are functionally and morphologically diverse. Inroads have been made in understanding the roles of distinct cortical interneuron subgroups, however, there are still many mechanisms to be worked out that may contribute to the development and maturation of different types of GABAergic cells. Moreover, altered GABAergic signaling may contribute to phenotypes of autism, schizophrenia and epilepsy. Specific Cre-driver lines have begun to parcel out the functions of unique interneuron subgroups. Despite the advances in mouse models, it is often difficult to efficiently study GABAergic cortical interneuron progenitors with molecular approaches in vivo. One important technique used to study the cell autonomous programming of these cells is transplantation of MGE cells into host cortices. These transplanted cells migrate extensively, differentiate, and functionally integrate. In addition, MGE cells can be efficiently transduced with lentivirus immediately prior to transplantation, allowing for a multitude of molecular approaches. Here we detail a protocol to efficiently transduce MGE cells before transplantation for in vivo analysis, using available Cre-driver lines and Cre-dependent expression vectors. This approach is advantageous because it combines precise genetic manipulation with the ability of these cells to disperse after transplantation, permitting greater cell-type specific resolution in vivo.

Since MGE cells have the unique ability to migrate and integrate when transplanted into a host neocortex 16, they provide an excellent model system for genetic manipulation before in vivo studies. Herein, we show how one can isolate MGE tissue from E13.5 embryos (Figures 1 and 2), which can then be transduced with lentiviruses either in vitro or in a rapid manner before transplantation for in vivo studies. Labeling MGE via lentiviruses has been performed before using...

Watch this Scientific Journal Video about Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence MGE Cells for In Vivo Studies at JoVE.com

Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence MGE Cells for In Vivo Studies

GABAergic cortical interneuron progenitors disperse, develop and synaptically integrate into a host cortex after transplantation. These cells can be easily transduced before transplantation for in vivo studies of genetically modified GABAergic precursors. Here, we show viral labeling techniques to target specific interneuron subgroups using existing Cre lines and Cre-dependent reporters.

Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence (MGE) Cells for In Vivo Studies

GABAergic cortical interneurons, derived from the embryonic medial and caudal ganglionic eminences (MGE and CGE), are functionally and morphologically ...

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Research

JoVE Journal

Developmental Biology

11.4K Views.  University of California San Francisco. GABAergic cortical interneuron progenitors disperse, develop and synaptically integrate into a host cortex after transplantation. These cells can be easily transduced before transplantation for in vivo studies of genetically modified GABAergic precursors. Here, we show viral labeling techniques to target specific interneuron subgroups using existing Cre lines and Cre-dependent reporters.

Video: Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence MGE Cells for In Vivo Studies

Intracranial Injection of Adeno-associated Viral Vectors

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific subsets of cells in different brain regions both in vivo and in brain sections. Unlike the injection of fluorescent dyes, viral labeling offers targeting of individual cell types and is less expensive and time consuming than establishing transgenic mouse lines. In this technique, an adeno-associated viral (AAV) vector is injected intracranially using stereotaxic coordinates, a micropipette and an automated pump for precise delivery of AAV to the desired area with minimal damage to the surrounding tissue. Injection parameters can be tailored to individual experiments by adjusting the animal age at injection, injection location, volume of injection, rate of injection, AAV serotype and the promoter driving gene expression. Depending on the conditions chosen, virally-induced transgene expression can allow visualization of groups of cells, individual cells or fine cellular processes, down to the level of dendritic spines. The experiment shown here depicts an injection of double-stranded AAV expressing green fluorescent protein for the labeling of neurons and glia in the mouse primary visual cortex.

Here we present the intracranial injection of AAV vectors for fluorescent labeling of neurons and glia in the visual cortex.

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific ...

Neuroscience

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

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.

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.

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in vivo gene control of neocortical projection neuron morphology. This method is based on (1) in vitro lentiviral engineering of neuronal precursors as "test" and "control" cells, (2) their co-transplantation into wild-type brains, and (3) paired morphometric evaluation of their neuronal derivatives. Specifically, E12.5 pallial precursors from panneuronal, genetically labeled donors, are employed for this purpose. They are engineered to take advantage of selected promoters and tetON/OFF technology, and they are free-hand transplanted into neonatal lateral ventricles. Later, upon immunofluorescence profiling of recipient brains, silhouettes of transplanted neurons are fed into NeurphologyJ open source software, their morphometric parameters are extracted, and average length and branching index are calculated. Compared to other methods, this one offers three main advantages: it permits achieving of fine control of transgene expression at affordable costs, it only requires basic surgical skills, and it provides statistically reliable results upon analysis of a limited number of animals. Because of its design, however, it is not adequate to address non cell-autonomous control of neuroarchitecture. Moreover, it should be preferably used to investigate neurite morphology control after completion of neuronal migration. In its present formulation, this method is exquisitely tuned to investigate gene control of glutamatergic neocortical neuron architecture. Taking advantage of transgenic lines expressing EGFP in other specific neural cell types, it can be re-purposed to address gene control of their architecture.

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.

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in ...

Genetics

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or triple transgenic mice are generated in order to achieve selective expression of a reporter or effector gene (e.g., from the Rosa26 locus) in the desired spinal population. 2) Intraspinal injection of Cre-dependent recombinant adeno-associated virus (rAAV); here Cre-dependent AAV vectors coding for the reporter or effector gene of choice are injected into the spinal cord of mice expressing Cre recombinase in the desired neuronal subpopulation. This protocol describes how to generate Cre-dependent rAAV vectors and how to transduce neurons in the dorsal horn of the lumbar spinal cord segments L3-L5 with rAAVs. As the lumbar spinal segments L3-L5 are innervated by those peripheral sensory neurons that transmit sensory information from the hindlimbs, spontaneous behavior and responses to sensory tests applied to the hindlimb ipsilateral to the injection side can be analyzed in order to interrogate the function of the manipulated neurons in sensory processing. We provide examples of how this technique can be used to analyze genetically defined subsets of spinal neurons. The main advantages of virus-mediated transgene expression in Cre transgenic mice compared to classical reporter mouse-induced transgene expression are the following: 1) Different Cre-dependent rAAVs encoding various reporter or effector proteins can be injected into a single Cre transgenic line, thus overcoming the need to create several multiple transgenic mouse lines. 2) Intraspinal injection limits manipulation of Cre-expressing cells to the injection site and to the time after injection. The main disadvantages are: 1) Reporter gene expression from rAAVs is more variable. 2) Surgery is required to transduce the spinal neurons of interest. Which of the two methods is more appropriate depends on the neuron population and research question to be addressed.

Intraspinal injection of recombinase dependent recombinant adeno-associated virus (rAAV) can be used to manipulate any genetically labelled cell type in the spinal cord. Here we describe how to transduce neurons in the dorsal horn of the lumbar spinal cord. This technique enables functional interrogation of the manipulated neuron subtype.

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or ...

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

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative cell replacement therapies while also creating tools to study cell fate in situ. To date, it has been possible to obtain neurons via in vivo reprogramming, but the precise phenotype of these neurons or how they mature has not been analyzed in detail. In this protocol, we describe a more efficient conversion and cell-specific identification of the in vivo reprogrammed neurons, using an AAV-based viral vector system. We also provide a protocol for functional assessment of the reprogrammed cells&#8217; neuronal maturation. By injecting flip-excision (FLEX) vectors, containing the reprogramming and synapsin-driven reporter genes&#160;to specific cell types in the brain that serve as the target for cell reprogramming. This technique allows for the easy identification of newly reprogrammed neurons. Results show that the obtained reprogrammed neurons functionally mature over time, receive synaptic contacts and show electrophysiological properties of different types of interneurons. Using the transcription factors Ascl1, Lmx1a and Nurr1, the majority of the reprogrammed cells have properties of fast-spiking, parvalbumin-containing interneurons.

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.

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative ...

Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain

Cell therapy has long been an emerging treatment paradigm in experimental neurobiology. However, cell transplantation studies often rely on end-point measurements and can therefore only evaluate longitudinal changes of cell migration and survival to a limited extent. This paper provides a reliable, minimally invasive protocol to transplant and longitudinally track neural progenitor cells (NPCs) in the adult mouse brain. Before transplantation, cells are transduced with a lentiviral vector comprising a bioluminescent (firefly-luciferase) and fluorescent (green fluorescent protein [GFP]) reporter. The NPCs are transplanted into the right cortical hemisphere using stereotaxic injections in the sensorimotor cortex. Following transplantation, grafted cells were detected through the intact skull for up to five weeks (at days 0, 3, 14, 21, 35) with a resolution limit of 6,000 cells using in vivo bioluminescence imaging. Subsequently, the transplanted cells are identified in histological brain sections and further characterized with immunofluorescence. Thus, this protocol provides a valuable tool to transplant, track, quantify, and characterize cells in the mouse brain.

Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain

We describe the intraparenchymal transplantation of human neural progenitor cells transduced with a dual reporter vector expressing luciferase-green fluorescent protein (GFP) in the mouse brain. After transplantation, the luciferase signal is repeatedly measured using in vivo bioluminescence and GFP-expressing grafted cells identified in brain sections using fluorescence microscopy.

Cell therapy has long been an emerging treatment paradigm in experimental neurobiology. However, cell transplantation studies often rely on end-point ...

In Vivo Visualization of the Rat Trigeminal Ganglion With Calcium Indicators

In-Vivo Calcium Imaging of Sensory Neurons in the Rat Trigeminal Ganglion

Genetically encoded calcium indicators (GECIs) enable imaging techniques to monitor changes in intracellular calcium in targeted cell populations. Their large signal-to-noise ratio makes GECIs a powerful tool for detecting stimulus-evoked activity in sensory neurons. GECIs facilitate population-level analysis of stimulus encoding with the number of neurons that can be studied simultaneously. This population encoding is most appropriately done in vivo. Dorsal root ganglia (DRG), which house the soma of sensory neurons innervating somatic and visceral structures below the neck, are used most extensively for in vivo imaging because these structures are accessed relatively easily. More recently, this technique was used in mice to study sensory neurons in the trigeminal ganglion (TG) that innervate oral and craniofacial structures. There are many reasons to study TG in addition to DRG, including the long list of pain syndromes specific to oral and craniofacial structures that appear to reflect changes in sensory neuron activity, such as trigeminal neuralgia. Mice are used most extensively in the study of DRG and TG neurons because of the availability of genetic tools. However, with differences in size, ease of handling, and potentially important species differences, there are reasons to study rat rather than mouse TG neurons. Thus, we developed an approach for imaging rat TG neurons in vivo. We injected neonatal pups (p2) intraperitoneally with an AAV encoding GCaMP6s, resulting in &gt;90% infection of both TG and DRG neurons. TG was visualized in the adult following craniotomy and decortication, and changes in GCaMP6s fluorescence were monitored in TG neurons following stimulation of mandibular and maxillary regions of the face. We confirmed that increases in fluorescence were stimulus-evoked with peripheral nerve block. While this approach has many potential uses, we are using it to characterize the subpopulation(s) of TG neurons changed following peripheral nerve injury.

Author Spotlight: Exploring Peripheral Mechanisms of Neuropathic Pain in Trigeminal Nerve Injury 

Genetically encoded calcium indicators (GECI) enable a robust, population-level analysis of sensory neuron signaling. Here, we have developed a novel approach that allows for in vivo GECI visualization of rat trigeminal ganglia neuron activity.

Genetically encoded calcium indicators (GECIs) enable imaging techniques to monitor changes in intracellular calcium in targeted cell populations. Their ...

Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence (MGE) Cells for In Vivo Studies

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Chapters in this video

Intracranial Injection of Adeno-associated Viral Vectors

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

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

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

Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain

In-Vivo Calcium Imaging of Sensory Neurons in the Rat Trigeminal Ganglion