We thank Mihn Duc Do for his contribution to early setting up of this procedure.

All methods and procedures described here have been approved by the SISSA Organismo preposto al Benessere Animale (SISSA IACUC).
1. Generation of engineered &#34;green&#34; progenitor pools
<ol>
	<li>Preparation of the &#34;green&#34; pool
	<ol>
		<li>Mate a wild-type CD1 female with a MtaptEGFP/+&#160;founder16. Euthanize the pregnant dam by cervical dislocation at 12.5 days post-coitum (day 0 is determined by vaginal plug inspection) and harvest the embryonic day 12.5 (E12.5) embryos. Set them in individual wells of a 24 multiwell plate in cold PBS solution.</li>
		<li>Quickly genotype the embryos by visual inspection under a blue light lamp, checking for green fluorescence emission in the brains.</li>
		<li>Place the &#34;green&#34; embryos into a 10 cm diameter Petri dish filled with cold 1x PBS with 0.6% glucose and transfer it to under a stereomicroscope.</li>
		<li>Cut the embryo heads using standard scissors and extract the telencephalon from them by means of #3 and #5 forceps.</li>
		<li>Separate the two telencephalic vesicles and dissect out the neocortices using forceps; make sure to remove the hippocampus and the basal ganglia17.</li>
		<li>Collect neocortices by transferring them with a P1000 pipette into a 1.5 mL tube kept on ice.</li>
		<li>Let the dissected neocortices settle to the bottom of the tube.</li>
		<li>Aspirate the supernatant as much as possible.</li>
		<li>Resuspend the neocortices in 400 &#181;L of fresh proliferative medium [DMEM-F12, 1x Glutamax, 1X N2, 1 mg/mL bovine serum albumin (BSA), 0.6% glucose, 2 &#181;g/mL heparin, 20 ng/mL basic fibroblast growth factor (bFGF), 20 ng/mL epidermal growth factor (EGF), 1X pen-strep, and 10 pg/mL amphotericin B].</li>
		<li>Gently pipette the neocortices up and down, sequentially with P1000, P200, and P20 tips, 4x per tip, to obtain a cloudy cell suspension. 
		NOTE: E12.5 neocortical tissue is very soft; dissociating it to single cells does not require enzymatic digestion at all; repeated pipetting is sufficient.</li>
		<li>Let the meninges and residual neocortical clumps settle down for 2 min.</li>
		<li>Transfer 150 &#181;L from the top of the cell suspension (mainly containing single cells) into a new 1.5 mL sterile tube.</li>
		<li>Add 150 &#181;L of fresh proliferative medium and repeat steps 1.1.10-1.1.12 until no more tissue clumps are evident. While doing this, omit the P1000 pipetting step (in step 1.1.10). 
		NOTE: Three iteration of steps 1.1.10-1.1.12 are usually sufficient to achieve full tissue dissociation.</li>
		<li>Count cells (&#34;green&#34; pool) with the trypan blue exclusion method in a B&#252;rker chamber17 and add fresh proliferative medium to adjust the concentration to 103 cells/&#181;L. 
		NOTE: Steps 1.1.7-1.1.14 must be performed under a sterile laminar flow hood disinfected by 70% ethanol and kept ventilated for at least 20 min.</li>
	</ol>
	</li>
	<li>Engineering the &#34;green&#34; sub-pools
	<ol>
		<li>Subdivide the &#34;green&#34; pool into two sub-pools in two distinct 1.5 mL tubes for lentiviral infection.</li>
		<li>Infect the sub-pools independently with the two dedicated lentiviral mixes. Each mix contains &#34;red label&#34; virus (Pgk1_mCherry) or &#34;black&#34; virus (pCAG_lacZ) as a control, as well as viruses for tetOFF driven transgene (Pgk1p_tTA and TRE_transgene) or control (Pgk1p_tTA and TRE_PLAP) expression. 
		CAUTION: Manipulate lentiviruses in a BLS-2 environment with all the protective measurements prescribed by applicable rules and laws. 
		NOTE: Each lentivirus must be administered at a multiplicity of infection (MOI) of 8, which (as previously shown14) is sufficient to transduce the vast majority of neural cells in such experimental conditions (Figure 1). The lentivirus harboring tet transactivators can be built by employing different neuronal promoters driving tTA/rtTA expression (e.g., pT&#945;1, pSyn, pCaMKII, pGAD1, etc.) (Figure 2). The MOI is the ratio of (a) the number of infectious viral particles delivered to (b) the number of their target cells. Here, the former (a) is calculated according to the conventional procedure described elsewhere14, the latter (b) is simply evaluated using a B&#252;rker chamber.</li>
		<li>Plate 600,000 cells of each infected sub-pool per well of a 12 multiwell plate, in 600 &#181;L of proliferative medium with 2 &#181;g/mL of doxycycline. 
		NOTE: Here, wells are not pretreated with poly-lysine, which prevents neural cell attachment to their bottoms.</li>
		<li>Transfer cells to the incubator in 5% CO2 at 37 &#176;C.</li>
		<li>After 2 days, to assess the efficiency of infection and the differential marking of the engineered pools, inspect the cells under a conventional fluorescent microscope. The two sub-pools must appear as suspensions of small neurospheres, homogenously expressing the red fluorescent protein (&#34;control&#34; sample) or not (&#34;test&#34; sample). Within these neurospheres, the MtaptEGFP transgene is active limited to a minority of cells (post-mitotic neurons) and silent in the majority of them (proliferating precursors) (Figure 3).</li>
	</ol>
	</li>
</ol>
2. Setting up of the surgical instruments and the operating area
<ol>
	<li>Preparing borosilicate needles
	<ol>
		<li>Use the borosilicate glass capillaries with external and internal diameter equaling 1.5 mm and 1.12 mm, respectively.</li>
		<li>Pull the capillary with a P 1000 puller.</li>
		<li>Set up the pulling program. Typical program parameters are: heat = 614; vel = 60; time = 1; pressure = 600. They must be adjusted according to the puller model and the heating resistor employed.</li>
		<li>Place the capillary into the puller holders and tighten them.</li>
		<li>Start the program and take the two pulled microcapillaries.</li>
		<li>Cut the capillary tip by hand, with a scalpel, under the stereomicroscope to obtain a tip with an external diameter of ~200-250 &#181;m.</li>
		<li>Place the capillaries on a modelling clay (e.g., plasticine) support in a closed Petri dish and transfer it to under the hood.</li>
	</ol>
	</li>
	<li>Setting up the hood and preparing the cell tracer solution
	<ol>
		<li>Clean up the horizontal flow hood carefully and disinfect it using 70% ethanol.</li>
		<li>Disinfect optical fibers using 70% ethanol and place them under the hood.</li>
		<li>Mix 10 &#181;L of 125 mM EGTA solution and 10 &#181;L of Fast Green to prepare the &#34;cell tracer solution&#34; and place it under the hood.</li>
		<li>Cut small pieces (4 cm x 2 cm of size) of laboratory sealing film for cell suspension spotting.</li>
		<li>Prepare a solution of 1 mg/mL sterile doxycycline and aspirate it into a 0.3 mL syringe.</li>
		<li>Switch on the hood and keep the laminar flow on for 20 min before the operation.</li>
	</ol>
	</li>
	<li>Assembling the injection tube
	<ol>
		<li>Take a hard-plastic mouthpiece, two latex tubes (each about 30 cm long) and a capillary holder from an aspirator tube assembly kit for calibrated microcapillary pipettes.</li>
		<li>Fix the hard-plastic mouthpiece to one end of a latex tube.</li>
		<li>Fix the capillary holder to one end of another latex tube.</li>
		<li>Connect the free ends of the two latex tubes with a 0.45 &#181;m sterile filter, as a barrier against operator germs.</li>
		<li>Place the resulting aspirator tube assembly on a modelling clay holder to be kept under the hood.</li>
	</ol>
	</li>
</ol>
3. Cell mix and intraventricular transplantation
<ol>
	<li>Mixing &#34;test&#34; and &#34;control&#34; green cell sub-pools
	<ol>
		<li>Collect &#34;test&#34; and &#34;control&#34; neurospheres (see step 1.2.5) in separate 1.5 mL tubes.</li>
		<li>Centrifuge neurospheres suspension at 200 g for 5 min.</li>
		<li>Collect and discard the supernatant, avoiding disturbance of the cell pellet.</li>
		<li>Gently resuspend neurospheres in 500 &#181;L of 1x PBS.</li>
		<li>Centrifuge them at 200 g for 1 min.</li>
		<li>Remove the supernatant.</li>
		<li>Repeat steps 3.1.4-3.1.6 two more times.</li>
		<li>Gently resuspend spheres in 200 &#181;L of 2x Trypsin solution (2x Trypsin, 1x PBS) and pipette cell pellet up and down 4x-5x. 
		NOTE: Pay attention not to make air bubbles.</li>
		<li>Leave cells in the incubator for 5 min at 37 &#176;C to achieve a single-cell suspension.</li>
		<li>Block Trypsin with 200 &#181;L of Trypsin inhibitor solution (DMEM-F12, 10 &#181;g/mL DNAse I, 0.28 mg/mL Trypsin inhibitor) by pipetting up and down 4x-5x.</li>
		<li>Centrifuge cells at 200 x g for 5 min and resuspend them in 1 mL of fresh proliferative medium (DMEM-F12, 1x Glutamax, 1x N2, 1 mg/mL BSA, 0.6% glucose, 2 &#181;g/mL heparin, 20 ng/mL bFGF, 20 ng/mL EGF, 1x pen-strep, 10 pg/mL amphotericin B).</li>
		<li>Count cells of the two pools with trypan blue exclusion method in a B&#252;rker chamber.</li>
		<li>Adjust their concentration to 100,000 cells/&#181;L in proliferative medium.</li>
		<li>Mix 1:1 &#34;test&#34; cells with &#34;control&#34; ones.</li>
		<li>Check the resulting mix under a fluorescence microscope (objective magnification: 10x) to ensure that the mix is a single-cell suspension of the two pools (Figure 3C).</li>
		<li>Place the mix on ice under the hood.</li>
	</ol>
	</li>
	<li>Intraventricular transplantation
	<ol>
		<li>Prepare the cage with the mother and the P0 pups on a table far away from the surgical operatory area.</li>
		<li>Place a recovery cage on a cart close to the hood.</li>
		<li>Put a mixture of sawdust taken from the mother&#39;s cage on the bottom of the recovery cage and place it under a lamp.</li>
		<li>Aside, set an ice box covered by an aluminum foil for the anesthesia of P0 pups.</li>
		<li>Just prior to transplantation, add 1/10 volume of cell tracer solution (from step 2.2.3) to 1 volume of cell suspension (from step 3.1.16) and spot 3 &#181;L of the resulting &#34;injection mix&#34; on a piece of laboratory sealing film.</li>
		<li>Place the pup on the cool aluminum foil for 1 min and check that it is fully anesthetized. 
		NOTE: To confirm anesthesia, gently squeeze a paw and monitor the pup for lack of movements, while still breathing.</li>
		<li>Meanwhile, aspirate the 3 &#181;L of injection mix (from step 3.2.5) into the glass capillary.</li>
		<li>Gently wipe the head of the anesthetized pup with 70% ethanol.</li>
		<li>Place the pup&#39;s chin on the tip of the optical fiber to clearly identify the cortical hemispheres. 
		NOTE: At P0-P1, the head skin is very thin, allowing for straightforward visual identification of the hemispheres just above the eyeballs and behind the olfactory bulbs.</li>
		<li>Keep the capillary (loaded as described in step 3.2.7) in either one of the two mid-parasagittal planes (left or right), above the olfactory bulb, and puncture the forehead skin at its intersection with the frontal plane containing the eyeballs centers. Pay attention not to damage blood vessels. Enter the capillary into the frontal cortex and access the ventricular cavity (Figure 4A).</li>
		<li>To prevent accidental damage of the ganglionic eminence, rotate the needle laterally by 30 degrees, pointing its tip caudal-medial-ward (Figure 4B).</li>
		<li>Gently inject the cells into the ventricular cavity and monitor their diffusion by means of Fast Green (Figure 4C).</li>
		<li>Wait 5-10 s.</li>
		<li>Remove the glass needle taking care not to aspirate the cell suspension and discard it in a suitable disposal bin.</li>
		<li>Before pup recovery from anesthesia inject 150 &#181;L of doxycycline solution intraperitoneally to keep transgene expression still off after the transplantation.</li>
		<li>After the injection, place the pup immediately under the lamp for recovery.</li>
		<li>Leave the pups under the lamp for 5-10 min and, once fully awake, place them in the cage with the mother.</li>
		<li>Observe the operated pups for 2-3 h, to ensure that the mother accepts them.</li>
		<li>Supply the cage with drinking water containing 0.5 mg/mL doxycycline and 50 g/L sucrose to maintain transgene expression off.</li>
		<li>Replace the doxycycline-containing water by doxy-free water 4 days after transplantation, thus activating the transgene in post-mitotic neurons. Keep mice in a doxy-free regime for 6 days and euthanize them at P10 by carbon dioxide inhalation followed by decapitation.</li>
	</ol>
	</li>
</ol>
4. Analysis of transplanted brains
<ol>
	<li>Histology and immunofluorescence
	<ol>
		<li>Euthanize the transplanted pups 10 days after cell transplantation by carbon dioxide inhalation followed by decapitation. Fix brains of euthanized pups in 4% paraformaldehyde (PFA) at 4 &#176;C overnight. 
		CAUTION: PFA is highly toxic; handle it with care, strictly complying with manufacturer&#39;s prescriptions, under a chemical fume hood; discard PFA solution residual in a labeled waste container.</li>
		<li>Remove the PFA solution and replace it with 30% sucrose solution.</li>
		<li>Leave the brains at 4 &#176;C or until they sink to the vial bottom.</li>
		<li>Transfer the brains into a disposable embedding mold approximately half-filled with cryo-inclusion medium.</li>
		<li>Freeze the included brains at -80 &#176;C.</li>
		<li>Cut 60 &#181;m thick coronal sections using a cryostat. 
		NOTE: Avoid employing thinner sections, as this may result in unacceptable loss of information regarding the whole architecture of single neurons.</li>
		<li>Process sections for immunofluorescence18,19, using anti-GFP [1:400] and anti-RFP (mCherry) [1:500] antibodies. Counterstain nuclei with 1 mg/mL DAPI solution [1:200].</li>
	</ol>
	</li>
	<li>Morphometry
	<ol>
		<li>Set working parameters of the confocal microscope as follows: z-stack height = 40 &#181;m; step = 2 &#181;m.</li>
		<li>Collect pictures of immunoassayed slices selecting GFP positive neuron-rich photographic fields, blind of RFP signal distribution.</li>
		<li>Export the images as .nd2 files.</li>
		<li>Generate maximum Z-projections of them as .tiff files (Figure 5A).</li>
		<li>To allow for sub-sequential neuronal skeletonization blind to cells genotype, hide the red signal. To do so, open each .tiff file by a suitable software and add an adjustment layer. Select &#34;levels&#34;, &#34;red&#34;, then set &#34;output&#34; to zero. Save the file as such. 
		NOTE: Skeletonization is subsequently performed by a new operator who had no previous access to original plain red files.</li>
		<li>For each hidden red file, add a drawing layer to the primary image and select the pencil tool (white color). Trace the soma on the basis of GFP signal, then trace the neurites. 
		NOTE: the pencil tool may be set at 40 pixels for the soma and at three pixels for neurites.</li>
		<li>Save the multilayer file including neuronal silhouettes (Figure 5B). 
		NOTE: Subsequent analysis of neuronal skeleton can be performed by either operator.</li>
		<li>Create a new 1024 x 1024 16-bit grayscale file with black background, one per neuron.</li>
		<li>Copy and paste single neuronal silhouettes from the primary image to the new grayscale file. Get back to multilayer file and switch off the adjustment layer to unveil neuronal genotypes. Save the 16-bit grayscale file annotating the corresponding neuronal genotype.</li>
		<li>Import grayscale images in ImageJ one by one and analyze skeletons by NeurphologyJ plugin of ImageJ software20 (Figure 5C).</li>
		<li>Copy NeurphologyJ primary data (neurite_length, attachment_points and endpoints; for each, take &#34;Total Area&#34; values), paste them into a spreadsheet and employ them to compute secondary morphometric parameters (average neurite length, branching index) (Figure 5D).</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Specific aspects/steps of this procedure are critical and require special attention. First, (a) operators must be adequately pretrained to safely manipulate lentiviruses in a BSL-2 compliant lab environment. Second, (b) prior to mix &#34;test&#34; and &#34;control&#34; neural preparations, it is mandatory to carefully wash the two corresponding neurosphere suspensions as described, in order to prevent any delayed cross-infection of the two preparations due to unwanted lentiviral carry over. Third, (c) while transplanting cells, care must be placed while targeting the ventricular cavity; in this respect, the Fast Green tracer included into the cell suspension is of substantial help. Additionally, (d) to prevent cannibalism, transplanted pups must be re-transferred to the mother only after their full recovery from anesthesia (usually 10 min is sufficient). (e) Cryo-sectioning of the transplanted tissue should be performed at 60 &#181;m; in fact, this thickness simultaneously allows an easy antibody penetration into the tissue and limits loss of information originating from artifactual neuron fragmentation. Finally, (f) to prevent any unintentional bias in data evaluation, we emphasize that neuron skeletonization must be performed by an operator blind of red channel.
The protocol described here refers to GOF gene manipulations. Alternatively, &#34;test&#34; neurons may be engineered to downregulate GOI function by means of lentiviruses encoding for RNAi effectors. Next, we usually employ mCherry to label &#34;control&#34; neural cells; however, the mCherry/&#34;control&#34; and LacZ/&#34;test&#34; scheme may be obviously inverted. Last, the protocol described here refers to intraventricular cell injections; &#34;test&#34; and &#34;control&#34; neurons may be alternatively co-injected into the neural parenchima21.
Despite its advantages, this method has two main limits. While allowing to investigate cell-autonomous gene control of neuroarchitecture, it does not apply to environmental control of it. Moreover, as transplanted neuronal precursors migrate from the ventricular aspect of the cortical wall to their final laminar location after birth (i.e. within a non-physiological timeframe), this method should be preferably employed to model neuroarchitectonics control after migration completion.
Last but not least, special attention should be paid to critical interpretation of results. In particular, if the X gene subject of investigation reduces the morphological complexity of &#34;test&#34; neurons, this might not specifically reflect its genuine impact on neuron architecture. Rather, such phenomenon might be a non-specific index of neuronal damage elicited by X overexpression. To address this issue, it may be useful to compare local densities of transplanted, &#34;test&#34; and &#34;control&#34; neurons, then look for possible numerical shrinkage of the &#34;test&#34; population, as an index of neuronal suffering induced by X [this shrinkage should be even more pronounced upon further delayed analysis of transplanted brains]. In such cases, lowering the overepression level of the X gene or moving to a loss-of-function design might fix the issue.
Our method adds to a wide array of technologies employed to investigate gene control of neuronal morphology in vivo1,2,3,4,7,8,9,10,11,12. As recalled in the introduction, these technologies rely on a variety of ad hoc genetic manipulations aimed to perturb GOI expression levels and ease the extraction of morphological information from the biological samples. These technologies usually allow accurate dissection of this control; however, they require not valuable experimental skills and financial resources. In this respect, the method offers three key advantages. It allows an accurate control of GOI expression levels comparable to those peculiar to transgenic models; however, in the absence of mutant colony maintenance costs. It does not require sophisticated experimental (surgical) skills. It often achieves statistical significance of results upon analysis of a relatively limited number of animals.

Here we describe a simple method we developed to dissect in vivo gene control of neuronal cytoarchitecture. Based on in vitro engineering of neuronal precursors, their transplantation into neonatal brain and paired morphometric evaluation of &#34;test&#34; and &#34;control&#34; cells, it allows to unveil functional implications of test genes in fine control of neuronal morphology in a fast and affordable way. To investigate in vivo&#160;gene control of neuronal architecture, three key technical issues must be addressed: (1) achieving an adequately patterned gene-of-interest (GOI) expression and an accurate quantitative control of it; (2) obtaining a properly segmented visualization of distinct neuronal silhouettes; (3) eliciting statistical significance of results while employing a limited number of animals.
When available, mouse mutant lines harboring tetracycline (tet)-controlled transgenes may be the best tool to address the first issue1. Alternatively, somatic transgenesis may be employed. In such cases, the transgene is delivered via electroporation2 or viral transduction3. Next, it is retained as an episome (e.g., upon standard electroporation4), or it is integrated into the genome (randomly, via retroviral integrase5; or in a defined location, via CRISPR-promoted homologous recombination (SLENDR)6).
Second, neuronal silhouette visualization may be achieved via (a) sparse uniform labeling or (b) dense differential labeling. As for sparse labeling, advanced Golgi-like methodologies may be employed7, selected neuronal minisets can be filled by biocytin8, and salt-and-pepper labeling can be obtained thanks to a sparsely expressed transgene. Such a transgene may display variegated transcription (Thy-EGFP)9 or may be activated by stochastic recombination (MORF)10. As for (b), state of art strategies include Cre-mediated stochastic recombination within a multi-floxed fluoroprotein transgene array (Brainbow)11, as well as piggyBac-transposase-driven genomic integration of fluoroprotein genes, previously delivered via somatic transgenesis (CLoNE)12.
As for the third issue, the morphometric outcome is often affected by a large random variability, originating from inter-animal differences and cell-injection contingencies. Because of this, a large number of animals is usually employed to achieve the statistical power necessary to assess GOI morphometric activity.
The approaches previously described often rely on advanced technical skills and require conspicuous financial resources, which may limit their diffusion within the scientific community. To circumvent these issues, we conceived an easy and straightforward pipeline to dissect gene control of neuroarchitecture in vivo in a fast and affordable way. This is inspired by a similar co-transplantation design previously developed for fast in vivo evaluation of antiblastic transgene activity13.
Specifically, it is thought that the co-transplantation of in vitro engineered &#34;green&#34; neural precursors (&#34;test&#34; and &#34;control&#34; cells) into a &#34;black&#34; recipient neonatal brain can simultaneously fix the three key issues listed above. In fact, in vitro lentiviral engineering of precursors, in well-controlled conditions, allows maintenance of variability of neuronal transgene expression at a minimum, far below that usually associated with in vivo somatic manipulations (performed previously14,15 and in our unpublished results). The resulting accurate control of gene expression is comparable with that achieved by tet-controlled transgenic models. However, costs of this procedure are far below those originating from maintenance of a transgenic mouse line. Next, free-hand cell injection is easy and requires minimal training. Moreover, the amount of labeled precursors injected into each brain can be easily tuned to achieve a sufficient cumulative number of sparsely distributed precursors, while also keeping the total number of transplanted animals at a minimum. Last but not least, co-injection of differently fluoro-labeled, &#34;test&#34; and &#34;control&#34; precursors and subsequent pairwise statistical analysis of the results counteract the effects of inter-animal experimental variability, allowing the reaching of statistical significance of results, even upon the analysis of a limited number of individuals13.
It should be emphasized that, albeit fast and cheap, this method has two main limitations. First, it is designed to investigate cell-autonomous gene control of neuronal architecture, and it is not appropriate to address environmental control. Second, as transplanted neuronal precursors reach their final location by a heterochronic schedule, this method is preferable to model neuroarchitectonics control occurring past migration completion.

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in house&lt;/em&gt;</td><td></td><td>store at -20 &amp;#176;C</td></tr><tr><td>Plasmid #408 [LV_Ppgk1p_rtTA(M2)]</td><td>built &lt;em&gt;in house&lt;/em&gt;</td><td></td><td>store at -20 &amp;#176;C</td></tr><tr><td>Plasmid #484 [LV_lacZ]</td><td>Addgene</td><td>12108</td><td>store at -20 &amp;#176;C</td></tr><tr><td>Plasmid #529 [LV_Pgk1p_mCherry]</td><td>built &lt;em&gt;in house&lt;/em&gt;</td><td></td><td>store at -20 &amp;#176;C</td></tr><tr><td>Plasmid #730 [LV_pSyn_rtTA(M2)]</td><td>built &lt;em&gt;in house&lt;/em&gt;</td><td></td><td>store at -20 &amp;#176;C</td></tr><tr><td>Scalpel</td><td>Braun</td><td>BB515</td><td>store at RT</td></tr><tr><td>Steromicroscope</td><td>Leica</td><td>MZ6</td><td>store at RT</td></tr><tr><td>Trypan blue</td><td>Gibco</td><td>15250-061</td><td>store at RT</td></tr><tr><td>Trypsin</td><td>Gibco</td><td>15400-054</td><td>store at -20 &amp;#176;C</td></tr><tr><td>Trypsin inhibitor</td><td>Sigma</td><td>T6522</td><td>store at -20 &amp;#176;C</td></tr></tbody></table>

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.

There are five primary datasets providing useful information about key aspects of the procedure, the first being (1) efficiency of neural precursors transduction and co-transduction by lentiviral vectors. (2) An example of key features of the promoters employed to drive the &#34;test gene&#34;. (3) An example of engineered cells ready for transplantation. (4) A cartoon including key procedural details of cell microinjection into the neonatal brain. (5) A synopsis of the whole morphometric pipeline.
As for (1), the capability of the prototypical lentiviral vectors delivered at different MOIs (2,4,8) to effectively transduce neocortical precursors was evaluated. In the first assay, neural precursors originating from early wild-type neocortices were infected by a lentiviral reporter, harboring the mCherry coding sequence (cds) under the control of the neuronogenic-lineage-specific pT&#945;1 promoter, at MOI = 2, 4, 8, then allowed to differentiate. Co-immunoprofiling of their progenies for mCherry and the pan-neuronal marker Tubb3 showed that neurons were effectively transduced at frequencies of 64%, 69%, and 78%, respectively (Figure 1A). In a second assay, neocortical precursors were acutely coinfected by two lentiviral reporters, expressing EGFP and mCherry, under the control of the constitutive pPgk1 promoter, at MOIs = (2,2), (4,4), and (8,8). A few days later, co-immunoprofiling of their derivatives showed that the ratio between EGFP+mCherry+ and EGFP+mCherry&#177; cells was 80%, 88%, and 93%, respectively (Figure 1B). These data suggest that, upon delivery of the engineering protocol envisaged for the present study, (a) the vast majority of neurons are transduced, and (b) almost all transduced neurons received the full lentiviral set needed for their characterization.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59242/59242fig1.jpg" /> 
Figure 1: Efficiency of neural precursor cells transduction by lentiviral vectors. Panels report an evaluation of the efficiency by which E12.5 neocortical precursor cells are transduced (or co-transduced) upon delivery of dedicated lentiviral reporters at different multiplicities of infection (MOIs). In the former case (A), cells were acutely infected with the lentivirus (LV) pT&#945;1-mCherry at MOI = 2, 4, 8, cultured in proliferative medium for 2 days, transferred to differentiative medium, and allowed to differentiate for 1 week. Upon fixation at day in vitro (DIV) 9, cultures were co-immunoprofiled for mCherry and the pan-neuronal marker Tubb3. mCherry and EGFP signals were detected by anti-RFP and anti-EGFP primary antibodies and revealed by Alexa-594- and Alexa-488-conjugated secondary antibodies, respectively. Finally, mCherry+Tubb3+/ mCherryTubb3+ ratios were calculated, averaged and plotted against the corresponding MOIs. In the latter case (B), cells were acutely infected with a 1:1 mix of two lentiviruses, encoding for the constitutively active pPgk-mCherry and pPgk-EGFP transgenes, at different MOIs (2, 4, 8 for each virus). Cells were cultured in proliferative medium for 4 days, trypsinized and, upon fixation, profiled for mCherry and EGFP immunofluorescence as above. Finally, mCherry+EGFP+/ mCherryEGFP+ ratios were calculated, averaged and plotted against the corresponding MOIs. Error bars represent S.E.M. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59242/59242fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
As for (2), pT&#945;1 and pSyn activity patterns can be inferred on the basis of expression profiles of fluoroprotein genes under their control. In this example, such control is direct in case of mCherry (Figure 2A), and indirect [i.e. mediated by a tetON second generation interface (Figure 2E)], in the case of EGFP (Figure 2B,C,D). Both promoters are specifically active within Tubb3+ postmitotic neurons (Figure 2B,C,D), but pT&#945;1 also fires in a subset of Ki67+ intermitotic (neuronogenic) precursors (Figure 2A). Here, to provide an idea of limited variability of promoter activity when cells are engineered according to these standard conditions (Figure 2E), pT&#945;1- and pSyn-driven EGFP expression levels within Tubb3+ neurons were quantified by Photoshop CS6 Histogram plugin. Then, raw data were normalized against medians and plotted against promoters (Figure 2F). Remarkably, single-cell fluorescence levels were densely clustered around medians: first and third quartiles equaled 0.86 and 1.16, as well as 0.89 and 1.12 in cases of pT&#945;1 and pSyn, respectively.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59242/59242fig2.jpg" /> 
Figure 2: Profiling of neural precursor type-specific promoters suitable to drive GOI overexpression. Panels refer to characterization of Tubulin alpha 1 (pT&#945;1) and Synapsin (pSyn) promoters, specifically active within the whole neuronal lineage and postmitotic neurons, respectively. Test were run in neocortical precursors harvested at different embryonic (En) ages, acutely infected with dedicated lentiviral mixes [pT&#945;1-mCherry in (A); pT&#945;1-rtTA, TREt-EGFP in (B); pSyn-rtTA, TREt-EGFP in (C,D); 2&#181;g/ml doxy ab initio], and cultured in proliferative medium (A), proliferative (2 days) followed by differentiative medium (B,C), or differentiative (D) medium. Upon fixation at day in vitro (DIV) n, postmitotic neurons were identified by &#945;Tubb3 and intermitotic precursors by &#945;Ki67 immunofluorescence; mCherry and EGFP were detected as shown in Figure 1. Signals were revealed as shown in Figure 1. Scale bars: 100 &#181;m in (A,B) and 50 &#181;m in (C,D). Shown are (E) dedicated lentiviral mixes used in this study with references to the figure panels. Shown is a (F) a scatter plot of pT&#945;1- and pSyn-driven EGFP signal in Tubb3+ cells referred to in (B) and (D), respectively. Boxed are cells falling between 25th and 75th percentiles; whiskers refer to 10th and 90th percentiles. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59242/59242fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
As for (3), 3 days after lentiviral transduction, MtaptEGFP/+ &#34;green&#34; neurospheres express the Pgk1p promoter-driven, mCherry red fluorescent protein (&#34;control&#34; spheres) or not (&#34;test&#34; spheres) (Figure 3A,B). All spheres are dissociated to single cells, ensuring there are no clumps lefts, and the corresponding suspensions are mixed 1:1. The resulting mix (Figure 3C) is placed on ice and used within 20 min for transplantation.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59242/59242fig3.jpg" /> 
Figure 3: Example of test (&#34;only green&#34;) and control (&#34;green-red&#34;) DIV2 neurospheres and cell suspension ready for transplantation. (A,B) These spheres were obtained from MtaptEGFP/+E 12.5 neocortical precursors, acutely infected by lentiviral mixes &#34;pCAG_lacZ, Pgk1p_tTA, TREt_GOI&#34; (A) and &#34;Pgk1p_mCherry, Pgk1p_tTA, TREt_PLAP&#34; (B), respectively, each LV at MOI = 8, and kept in proliferative medium. (C) Cell suspension was obtained by dissociating &#34;test&#34; and &#34;control&#34; neurospheres (A,B) to single cells and mixing them 1:1. Scale bars: 200 &#181;m in (A,B) and 100 &#181;m in (C). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59242/59242fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
As for (4), the procedure for cell injection into the neonatal brain includes three key steps. The capillary, placed on the mid-parasagittal plane, is entered into the lateral ventricular cavity via the frontal cortical wall (Figure 4A). Next, to prevent damage of the ganglionic eminence, the capillary is rotated 30&#176; within the horizontal plane, so that the tip points medially/caudally (Figure 4B). Last, the cell suspension is delicately ejected into the lateral ventricular cavity, where it forms an easily distinguishable, light-blue &#34;cloud&#34; (Figure 4C).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59242/59242fig4.jpg" /> 
Figure 4: Schematics of the three steps procedure employed for cell injection into the neonatal brain. (A) The capillary is entered into the lateral ventricle through the frontal neocortical wall. Then, (B) it is rotated medialward, to prevent damage of ganglionic eminence. Finally (C), the cell suspension is gently injected into the lateral ventricle, where it forms a light blue cloud. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59242/59242fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
As for (5), the analytical procedure includes four main steps. First, optical confocal sections of transplanted neuron-rich fields are flattened, according to MAX Z-projection modality so generating RGB .tiff files. Here, as an example, only &#34;green test&#34; neurons and &#34;yellow control&#34; neurons, originating from co-transplanted precursors, can be easily distinguished (it should be noted that mCherry may alternatively be used to label &#34;test&#34; neurons) (Figure 5A). Then, an idealized, skeletonized camera lucida version of the picture is generated by a suitable graphic software (see step 4.2.6) (Figure 5B) by an operator blind of red channel. Blindness of red channel is of paramount importance in order to prevent any unintentional bias in data evaluation. Next, single-neuronal silhouettes are fed into NeurphologyJ software as black-and-white pictures and values of the three primary parameters &#34;total number of exitpoints&#34; (attachment_points), &#34;total number of endpoints&#34; (endpoints) and &#34;total neurite length&#34; (neurite_length) are collected (Figure 5C). Finally, secondary parameters of each neuron are calculated, averaged and statistically evaluated by Excel software (Figure 5D).
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59242/59242fig5.jpg" /> 
Figure 5: Representative flowchart of morphometric analysis of transplanted brains. Upper row panels show: (A) a MAX z-projection .tiff image of medial neocortex, fixed 10 days after engineered cell transplantation (green = Mtapt-driven, neuron-restricted EGFP; red = mCherry, constitutively labeling &#34;control&#34; cells; blue = DAPI), the green channel signal, and (B) its skeletonized e-camera lucida rendering. Scale bars: 50 &#181;m. The bottom row includes: (C) primary morphometric parameters extracted from neuronal skeletons by NeurphologyJ software analysis (neurite_length as&#160;&#931;li, attachment_points as Nexit and endpoints as Nend) and (D) secondary parameters calculated on their basis. This figure has been modified from Chiola et al.21. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59242/59242fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies at JoVE.com

Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies

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 ...

intraventricular-transplantation-engineered-neuronal-precursors-for

Nanyang Technological University

Research

JoVE Journal

Genetics

5.7K Views.  SISSA. Het algemene doel van deze techniek is de beoordeling van het effect van genexpressie manipulaties op corticale neuronale architectuur in vivo. Ons eerste belangrijke voordeel van deze procedure ten opzichte van die gebaseerd op een neutrale elektroporatie, is de fijne controle van genexpressieniveaus. Specifiek, deze geïntegreerde pijpleiding maakt de in vivo evaluatie van defect, verheven door kleine veranderingen in genexpressie op de controle van neuronale cytoarchitectuur.Bovendi...

Video: Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies

Intraspinal Cell Transplantation for Targeting Cervical Ventral Horn in Amyotrophic Lateral Sclerosis and Traumatic Spinal Cord Injury

Respiratory compromise due to phrenic motor neuron loss is a debilitating consequence of a large proportion of human traumatic spinal cord injury (SCI) cases 1 and is the ultimate cause of death in patients with the motor neuron disorder, amyotrophic laterals sclerosis (ALS) 2.
ALS is a devastating neurological disorder that is characterized by relatively rapid degeneration of upper and lower motor neurons. Patients ultimately succumb to the disease on average 2-5 years following diagnosis because of respiratory paralysis due to loss of phrenic motor neuron innnervation of the diaphragm 3. The vast majority of cases are sporadic, while 10% are of the familial form. Approximately twenty percent of familial cases are linked to various point mutations in the Cu/Zn superoxide dismutase 1 (SOD1) gene on chromosome 21 4. Transgenic mice 4,5 and rats 6 carrying mutant human SOD1 genes (G93A, G37R, G86R, G85R) have been generated, and, despite the existence of other animal models of motor neuron loss, are currently the most highly used models of the disease.
Spinal cord injury (SCI) is a heterogeneous set of conditions resulting from physical trauma to the spinal cord, with functional outcome varying according to the type, location and severity of the injury 7. Nevertheless, approximately half of human SCI cases affect cervical regions, resulting in debilitating respiratory dysfunction due to phrenic motor neuron loss and injury to descending bulbospinal respiratory axons 1. A number of animal models of SCI have been developed, with the most commonly used and clinically-relevant being the contusion 8.
Transplantation of various classes of neural precursor cells (NPCs) is a promising therapeutic strategy for treatment of traumatic CNS injuries and neurodegeneration, including ALS and SCI, because of the ability to replace lost or dysfunctional CNS cell types, provide neuroprotection, and deliver gene factors of interest 9.
Animal models of both ALS and SCI can model many clinically-relevant aspects of these diseases, including phrenic motor neuron loss and consequent respiratory compromise 10,11. In order to evaluate the efficacy of NPC-based strategies on respiratory function in these animal models of ALS and SCI, cellular interventions must be specifically directed to regions containing therapeutically relevant targets such as phrenic motor neurons. We provide a detailed protocol for multi-segmental, intraspinal transplantation of NPCs into the cervical spinal cord ventral gray matter of neurodegenerative models such as SOD1G93A mice and rats, as well as spinal cord injured rats and mice 11.

Neural precursor transplantation is a promising strategy for protecting and/or replacing lost/dysfunctional cervical phrenic motor neurons in spinal cord injury (SCI) and the motor neuron disorder, amyotrophic laterals sclerosis (ALS). We provide a protocol for cell delivery to cervical spinal cord ventral horn in rodent models of ALS and SCI.

Respiratory compromise due to phrenic motor neuron loss is a debilitating consequence of a large proportion of human traumatic spinal cord injury (SCI) ...

Medicine

Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live animal &#8211;&#160;particularly in sites with unique physiological and architectural qualities such as the brain. In vitro and ectopic models cannot account for features such as vasculature, blood brain barrier, metabolism, drug delivery and toxicity, and a host of other relevant factors. Orthotopic models have their limitations too, but with proper technique tumor cells of interest can be accurately engrafted into tissue that most closely mimics conditions in the human brain. By employing methods that deliver precisely measured volumes to accurately defined locations at a consistent rate and pressure, mouse models of human brain tumors with predictable growth rates can be reproducibly created and are suitable for reliable analysis of various interventions. The protocol described here focuses on the technical details of designing and preparing for an intracranial injection, performing the surgery, and ensuring successful and reproducible tumor growth and provides starting points for a variety of conditions that can be customized for a range of different brain tumor models.

The brain is a unique site with qualities that are not well represented by in vitro or ectopic analyses. Orthotopic mouse models with reproducible location and growth characteristics can be reliably created with intracranial injections using a stereotaxic fixation instrument and a low pressure syringe pump.

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

Neuroscience

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

Bioengineering

Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory environment and the limited capacity for neurogenesis. We are developing&#160;a strategy to simultaneously address&#160;neuronal and axonal pathway loss within the damaged CNS. This manuscript presents the fabrication protocol for micro-tissue engineered neural networks (micro-TENNs), implantable constructs consisting of neurons and aligned axonal tracts spanning the extracellular matrix (ECM) lumen of a preformed hydrogel cylinder hundreds of microns in diameter that may extend centimeters in length. Neuronal aggregates are delimited to the extremes of the three-dimensional encasement and are spanned by axonal projections. Micro-TENNs are uniquely poised as a strategy for CNS reconstruction, emulating aspects of brain connectome cytoarchitecture and potentially providing means for network replacement. The neuronal aggregates may synapse with host tissue to form new functional relays to restore and/or modulate missing or damaged circuitry. These constructs may also act as pro-regenerative&#160;&#34;living scaffolds&#34; capable of exploiting developmental mechanisms for cell migration and axonal pathfinding, providing synergistic structural and soluble cues based on the state of regeneration. Micro-TENNs are fabricated by pouring liquid hydrogel into a cylindrical mold containing a longitudinally centered needle. Once the hydrogel has gelled, the needle is removed, leaving a hollow micro-column. An ECM solution is added to the lumen to provide an environment suitable for neuronal adhesion and axonal outgrowth. Dissociated neurons are mechanically aggregated for precise seeding within one or both ends of the micro-column. This methodology reliably produces self-contained miniature constructs with long-projecting axonal tracts that may recapitulate features of brain neuroanatomy. Synaptic immunolabeling and genetically encoded calcium indicators suggest that micro-TENNs possess extensive synaptic distribution and intrinsic electrical activity. Consequently, micro-TENNs represent a promising strategy for targeted neurosurgical reconstruction of brain pathways and may also be applied as biofidelic models to study neurobiological phenomena in vitro.

This manuscript details the fabrication of micro-tissue engineered neural networks: three-dimensional micron-sized constructs comprised of long aligned axonal tracts spanning aggregated neuronal population(s) encased in a tubular hydrogel. These living scaffolds can serve as functional relays to reconstruct or modulate neural circuitry or as biofidelic test-beds mimicking gray-white matter neuroanatomy.

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory ...

Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the roles of intrinsic vs. extrinsic mechanisms that regulate this differentiation process is a conundrum for all developmental neurobiologists. This issue is magnified for GABAergic interneurons, an incredibly heterogeneous cell population that is born from transient embryonic structures and undergo a protracted migratory phase to disperse throughout the telencephalon. To explore how different brain environments affect interneuron fate and maturation, we developed a protocol for harvesting fluorescently labeled immature interneuron precursors from specific brain regions in newborn mice (P0-P2). At this age, interneuron migration is nearly complete and these cells are residing in their final resting environments with relatively little synaptic integration. Following collection of single cell solutions via flow cytometry, these interneuron precursors are transplanted into P0-P2 wildtype postnatal pups. By performing both homotopic (e.g., cortex-to-cortex) or heterotopic (e.g., cortex-to-hippocampus) transplantations, one can assess how challenging immature interneurons in new brain environments affects their fate, maturation, and circuit integration. Brains can be harvested in adult mice and assayed with a wide variety of posthoc analysis on grafted cells, including immunohistochemical, electrophysiological and transcriptional profiling. This general approach provides investigators with a strategy to assay how distinct brain environments can influence numerous aspects of neuron development and identify if specific neuronal characteristics are primarily driven by hardwired genetic programs or environmental cues.

Challenging young neurons in new brain regions can reveal important insights into how the environment sculpts neuronal fate and maturation. This protocol describes a procedure to harvest interneuron precursors from specific brain regions and transplant them either homotopically or heterotopically into the brain of postnatal pups.

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the ...

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 ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of &#947;-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -&#160;MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice.

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

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 ...

Transplantation of Human Stem Cell-Derived GABAergic Neurons into the Early Postnatal Mouse Hippocampus to Mitigate Neurodevelopmental Disorders

A reduced number or dysfunction of inhibitory interneurons is a common contributor to neurodevelopmental disorders. Therefore, cell therapy using interneurons to replace or mitigate the effects of altered neuronal circuits is an attractive therapeutic avenue. To this end, more knowledge is needed about how human stem cell-derived GABAergic interneuron-like cells (hdINs) mature, integrate, and function over time in the host circuitry. Of particular importance in neurodevelopmental disorders is a better understanding of whether these processes in transplanted cells are affected by an evolving and maturing host brain. The present protocol describes a fast and highly efficient generation of hdINs from human embryonic stem cells based on the transgenic expression of the transcription factors Ascl1 and Dlx2. These neuronal precursors are transplanted unilaterally, after 7 days in vitro, to the hippocampus of neonatal 2-day-old mice. The transplanted neurons disperse in the ipsi- and contralateral hippocampus of a mouse model of cortical dysplasia-focal epilepsy syndrome and survive for up to 9 months after transplantation. This approach allows for investigating the cellular identity, integration, functionality, and therapeutic potential of transplanted interneurons over an extended time in developing healthy and diseased brains.

Transplantation of human pluripotent stem cell-derived GABAergic neurons generated by neuronal programming could be a potential treatment approach for neurodevelopmental disorders. This protocol describes the generation and transplantation of human stem cell-derived GABAergic neuronal precursors into the brains of neonatal mice, allowing long-term investigation of grafted neurons and evaluation of their therapeutic potential.

Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies

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