This work was supported by the Ministry of Science and Technology grants MOST104-2311-B-010-010-MY3, MOST106-2321-B-010-012, the National Health Research Institutes grant NHRI-EX106-10429NI, and&#160;the featured Areas Research Center Program grant from the Ministry of Education through Brain Research Center, National Yang-Ming University in Taiwan,&#160;and Postdoctoral Fellowship grants MOST106-2811-B-010-031 (S.-Y.C.), MOST105-2811-B-010-036 and MOST106-2811-B-010-030 (H.-Y.K.).

The animal protocols described here have been approved by the Animal Care and Use Committees of National Yang-Ming University.
1. Preparation of The Holder for Neonatal Pups in The Stereotaxic Apparatus
<ol>
	<li>Make the head tray: cut the bottom of a 1.5 mL centrifuge tube (15 mm long) into the shape that fits the head of neonatal pups by removing 1/5 of the wall of the tube.</li>
	<li>Take a pipette tip box with the right size that fits with the pedestal of stereotaxic apparatus, and remove the top cover. Affix the head tray prepared in step 1.1 and a tissue embedding cassette onto the base of tip tray with hot-melting adhesive. The height of the tissue embedding cassette is 7 mm, which is used to support the pup&#39;s neck and body in the head-fixed position.</li>
	<li>Place the whole setup in a standard stereotaxic apparatus.</li>
</ol>
2. Preparation of 30G Injection Stainless Steel Needles
<ol>
	<li>Pre-clean 30G syringe needles by soaking them in chloroform for 3 days.</li>
	<li>Use forceps to carefully and slowly pull the needle out from its polypropylene hub in a chemical hood.</li>
	<li>Wash the needles with absolute ethanol for 20 min followed by 70% ethanol rinses for 3 &#215; 10 min on a shaker at 50 rpm. Let the needles air dry, and store the cleaned needles in a clean box at room temperature (RT) until use.</li>
</ol>
3. Prepare an Adapter for Microinjection Tubing
<ol>
	<li>Connect the microliter syringe to a 30G injection needle with a PE10 polyethylene tube (PE10 tube). Prepare a PE20 polyethylene tubing (PE20 tube) adaptor for bridging the injection needle and the microliter syringe. The use of the adaptor is necessary, because the diameter of PE10 tube is much smaller than that of the needle of the 26G microliter syringe.</li>
	<li>Prepare the tubing for the microliter syringe: connect the pre-cleaned 30G injection needle with 5 cm of PE20 tube, and seal the junction with instant glue. This tubing adaptor is reusable. 
	NOTE: If the needle of microinjection syringe is 30G, the preparation (step 3.1) and usage (step 3.3) of this adaptor is not necessary.</li>
	<li>Connect the tubing adaptor (prepared in step 3.1) with a microliter syringe (10 &#181;L).</li>
	<li>Connect one end of PE10 tube (no longer than 60 cm) onto the 30G needle of the PE20 tubing adaptor. Mount a new 30G injection needle onto the other end of the PE10 tube.</li>
</ol>
4. Load the Microinjection Tube with Autoclaved Distilled Water, Dye and Viruses
<ol>
	<li>Remove the plunger of the microliter syringe. Use a 25G syringe to load the microliter syringe and its connected PE tube with autoclaved distilled water to remove air from the tubing.</li>
	<li>Place the plunger back to the microliter syringe, and push the plunger until 2 &#181;L of distilled water remains in the barrel. Do not let the volume of distilled water become lower than 2 &#181;L.</li>
	<li>Mount carefully the microliter syringe onto the micro flow rate syringe pump.</li>
	<li>Pipette small amounts of 0.1% fast green dye (prepared in 0.9% saline and filtered with 0.22 &#181;m filter) and the virus liquids to a piece of parafilm.</li>
	<li>Withdraw a small amount of air to make an air bubble visible at the junction between the 30G injection needle and PE10 tube followed by loading 0.7 &#181;L of filtered fast green into the tube to test the flow of fluids in the microinjection tubing.</li>
	<li>Withdraw another small amount of air to make a second air bubble followed by loading the virus liquids into the microinjection tubing.</li>
	<li>Attach and secure the 30G microinjection needle to the arm of stereotaxic apparatus.</li>
</ol>
5. Anesthesia of Neonatal Mice by Hypothermia
<ol>
	<li>Place the pup in a latex glove sleeve and immerse it in crushed ice up to the neck for 5 min.</li>
	<li>Pinch the pup&#39;s feet with forceps to make sure no withdrawal response of its feet.</li>
	<li>Place the pup with latex glove sleeve in the head tray and put some crushed ice around the latex sleeve to keep it cold for hypothermia anesthesia.</li>
	<li>Apply vet ointment on pup&#39;s eyes to prevent dryness while under anesthesia if possible.</li>
</ol>
6. Microinjection
<ol>
	<li>Prepare sterile surgery by wiping the stereotaxic instrument thoroughly with 70% ethanol. Sterilize the surgical instrument by immersing them in 70% ethanol.</li>
	<li>Scrub the pup&#39;s head with 70% ethanol. Locate the landmark lambda on the skull and mark the lambda with a marker pen. Aim the needle tip to the lambda, and set the anterior-posterior (AP) and medial-lateral (ML) coordinates as zero.</li>
	<li>Move the injection arm to the target site according to the X and Y coordinates of the target site. For the striatum of postnatal day (P) 2 pups, the coordinates are: AP, +2.4 mm anterior to the lambda; ML, &#177;1.0 mm lateral from the midline; dorsal-ventral (DV), &#8722;1.7 mm from the skull. Mark the position of fast green dye in PE10 tube with a pen.</li>
	<li>Bring down the 30G injection needle slowly to penetrate through the skin and skull, and then bring up the needle tip until it stops at the surface of the skull. Set the DV coordinate as zero.</li>
	<li>Bring down the 30G injection needle slowly until it reaches the DV coordinate of the target site. Wait for 1 min to allow the parenchyma resuming its normal shape. Run the microinjection program (100 nL/min).</li>
	<li>Make sure that the mark of fast green dye is moving in the PE tube to ensure the virus liquid is injected into the brain.</li>
	<li>Wait for 1 min after the termination of the microinjection, and then slowly and progressively bring up the needle to 1/2 height of DV depth within 30 s. After 30 s, slowly withdraw the needle from the pup&#39;s head.</li>
	<li>Repeat step 6.2 to 6.7 until the microinjections of all targeted sites are completed.</li>
</ol>
7. Post-surgical Recovery of The Pup
<ol>
	<li>Warm up the pup for 20 min in a 33 &#176;C incubator. Check the recovery of the pup from hypothermia anesthesia every 5 min until the pup has regained sufficient consciousness to maintain sternal recumbency.</li>
	<li>Return the pup back to the dam after the pup is fully recovered.</li>
</ol>

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M., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+investigation+of+neural+circuits+underlying+brain+disease+in+animal+models.">Optogenetic investigation of neural circuits underlying brain disease in animal models.</a> Nat. Rev. Neurosci. 13 (4), 251-266 (2012).</li><li>Roth, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DREADDs+for+neuroscientists.">DREADDs for neuroscientists.</a> Neuron. 89 (4), 683-694 (2016).</li><li>Knopfel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+optical+indicators+for+the+analysis+of+neuronal+circuits.">Genetically encoded optical indicators for the analysis of neuronal circuits.</a> Nat. Rev. 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The authors have nothing to disclose.

In the present study, we demonstrate a simple and reliable stereotaxic method for injecting AAV viruses into the striatum of neonatal mouse brains. We microinjected AAV-eGFP-Cre viruses into the striatum of Ai14 reporter mice at P2 and then analyzed the reporter gene expression at P14. We found AAV transduced GFP-positive cells throughout the striatum at rostrocaudal levels. Moreover, nearly all GFP-positive cells co-expressed the tdTomato reporter gene in striatal cells, suggesting a successful Cre-loxP DNA recombination induced by AAV-mediated expression of Cre activity. We further confirmed the efficacy of this approach by microinjections of AAV-eGFP-Cre into the striatum Foxp2fl/fl mice at P2. Double labeling showed that many AAVs-transduced GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting a successfully conditional deletion of Foxp2 gene in neonatal striatum. The volumes of microinjected AAVs are correlated with the size of the infected brain regions. With 200 nL of the injected volume, GFP-positive cells were found extensively throughout the neonatal striatum, and some scattered GFP-positive cells were also present in the cortex adjacent to the striatum. With 50 nL of the injected volume, GFP-positive cells were locally restricted to the neonatal striatum. Therefore, titrating the volumes of injected AAVs is important for optimal transduction of neuronal populations in specific brain regions. Moreover, the transduction rate is also determined by the titer of viral preparation. It is conceivable that precise targeting specific cell types can be achieved by using transgenic mice with different promoters.
There are several surgical tips for successful injecting AAVs to the interested brain regions. First, because of the small size of mouse pups and the difficulty in securing the position of the mouse head with hands during surgery, it is difficult to perform stereotaxic brain surgery in neonatal mice10. Although a previous study has demonstrated neonatal stereotaxic surgery, but it requires two people to perform the microinjection during the surgery11. We have overcome these problems by developing a custom-made head-fixed device that can firmly secure the pup head during brain surgery. The whole procedures can be performed by a single person. The brain sizes of neonatal mice are varied due to different genetic backgrounds, maternal care and the ages of pups. An advantage of our homemade device is that it allows a gentle but secured fixation of neonatal mouse head, which is essential for performing high precision stereotaxic brain surgery. Second, owing to the elasticity of the soft skin and the thin wall of skull, skin incision followed by skull drilling in the head is difficult to perform in neonatal pups. To overcome this problem, it is necessary to make a puncture of the skin and the skull with a needle tip prior to lowering the injection needle to the targeted brain region. Third, it is important to check the flow of visible dye in the PE tube to make sure the microinjection is in progression during injection. If the dye does not move in the PE tube, efforts should be taken to trouble shoot the problems, including a possible leakage of fluid in the tubing and clogged needles. Note that this method applies to the neonatal pups whose lambda are visible through the skin before P5. For pups older than P5, it may need an incision in the skin to expose the lambda. Note that light illumination onto P4 and P5 mouse heads may help locate the landmark lambda. Although we have not performed this technique of stereotaxic surgery in neonatal rats, we assume that the technique is applicable to rat pups with some modifications.
The onset and maximal expression of viruses carrying gene expression take several days or weeks after injections into the brains14,15. In the present study, we found that AAV-eGFP-Cre viruses-mediated Cre-loxP DNA recombination in neonatal striatal neurons occurred as early as 8 days after viral transduction. The previous study has reported that AAV-mediated Cre-dependent reporter activity can be detected in the olfactory bulb of newborn mouse pups as early as 3 days after the bulk injection of AAVs15. It is conceivable that the early onset of AAV-mediated genetic activity is an advantage for studying developmental events that continuously occur in the postnatal brains.
In summary, the neonatal stereotaxic technique that we developed is not only for conditionally delete genes in specific brain regions, but it is also amenable to inject pharmacological reagents and neuronal tracers to specific brain regions in neonatal mice. Although we only made microinjections into the neonatal striatum in the present study, in principle, our stereotaxic neonatal brain surgery can be applied to target specific brain regions if the coordinates of specific regions in neonatal brains are available. Given the variability in the size of neonatal mouse brains with different genetic backgrounds at different stages, it is essential to empirically determine the precise coordinates of the targeted sites by the investigators. Finally, by stereotaxic microinjections of AAVs expressing genetically modified optogenetic3 and chemogenetic proteins4 and neuronal activity indicators5 into specific brain regions, one may explore the role of neuronal activity on the postnatal development of the brain at the resolution of cell type- and circuit-specific levels.

Modern studies of the structure and function of the brain usually require genetic manipulation of specific genes in neuronal cells. To probe the functions of different genes, transgenic mice carrying mutant alleles, including knockout and knock-in alleles have been routinely generated. Stereotaxic brain surgery for adult rodents is a standard method to locally deliver drugs, viruses, tracers and other reagents to specific regions of rodent brains1,2. Applying the stereotaxic brain surgery to transgenic mice permits one to genetically manipulate the gene function and neuronal activity in specific neuronal populations of the mouse brain. The cell type-specific manipulation provides a powerful approach to decipher neuronal functions in complex neural circuits of the brain3,4,5.
Neural development of the nervous system begins at early embryonic stages, and the developmental processes continue after birth until the juvenile period. Postnatal maturation of the nervous system includes the precise synaptic wiring of neural circuits, which is essential for physiological and cognitive functions of the brain6. Therefore, studying developmental events that occur in neonatal time windows is important not only for understanding normal neural development, but it may also provide insights into the pathogenesis of neurodevelopmental and neuropsychiatric disorders7,8. Although the methods of stereotaxic brain surgery for adult rodents are readily available2,9, few protocols are available on the internet for stereotaxic brain surgery in neonatal mice10,11. In fact, stereotaxic microinjections of reagents into the brains of neonatal mouse pups are difficult, because the head of neonatal pup is too fragile to be fixed in the standard stereotaxic apparatus. Nonetheless, the application of stereotaxic brain surgery to transgenic mice is feasible for neonatal mice12. Here, we describe a simple method with a homemade setup to perform stereotaxic brain surgery in newborn mouse pups. We demonstrate that this technique allows one to conditionally delete floxed genes by microinjecting AAV-expressing Cre DNA recombinase into the striatum of reporter gene mice and conditionally floxed transgenic mice. This technique is also applicable to deliver reagents into the neonatal striatum of wild-type mice.

<table><tbody><tr><td>30G PrecisionGlide Needle</td><td>Becton Dickinson</td><td>REF 305106</td><td></td></tr><tr><td>Chloroform</td><td>JT Baker</td><td>9180-03</td><td></td></tr><tr><td>Hamilton MICROLITER Syringe</td><td>Hamilton&amp;nbsp;</td><td>80300</td><td>30G needle fit for PE10 tube; 26G needle needs a PE20 adaptor</td></tr><tr><td>Polyethylene tubing PE20</td><td>Becton Dickinson</td><td>427406</td><td></td></tr><tr><td>Polyethylene tubing PE10</td><td>Becton Dickinson</td><td>427401</td><td></td></tr><tr><td>Micro Flow Rate Syringe Pump</td><td>Longer Precision Pump Co.</td><td>TJ-2A (Controller) and L0107-2A (Drive Unit)</td><td></td></tr><tr><td>25G syringe</td><td>Becton Dickinson</td><td>REF 302105</td><td></td></tr><tr><td>Fast green</td><td>Sigma-Aldrich</td><td>F-7252</td><td>0.1%</td></tr><tr><td>Standard Stereotaxic Instruments</td><td>RWD Life Science</td><td>68037</td><td>Without using 68030 Mouse/Neonatal Rat Adaptor</td></tr><tr><td>Anti-FOXP2 antibody</td><td>Abcam</td><td>ab16046</td><td>Rabbit polyclonal to FOXP2, 1:4,000</td></tr><tr><td>Anti-RFP antibody</td><td>Abcam</td><td>ab65856</td><td>Mouse monoclonal to RFP, 1:1,000</td></tr><tr><td>BX63 microscope</td><td>Olympus</td><td>BX63</td><td></td></tr><tr><td>LSM 880 confocal microscope</td><td>Zeiss</td><td>LSM 880</td><td></td></tr><tr><td>Goat anti-rabbit conjugated Alexa fluor594</td><td>Jackson lmmunoReserch Laboratories Inc.</td><td>111-585-003</td><td></td></tr><tr><td>AAV9.hSynapsin.HI.eGFP-Cre.WPRE.SV40</td><td>Penn Vector Core</td><td>AV-9-PV1848</td><td>Lot # CS0987, 5.506x10&lt;sup&gt;13&lt;/sup&gt; (GC/mL)</td></tr><tr><td>AAV9.chicken actin-eGFP</td><td>AAV core, Institute of Biomedical Sciences, Academia Sinica, Taiwan</td><td>N/A</td><td>1x10&lt;sup&gt;14&lt;/sup&gt; (GC/ml)</td></tr><tr><td>B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J</td><td>The Jackson Labtorary&amp;nbsp;</td><td>007914</td><td>Ai14</td></tr><tr><td>B6(Cg)-Foxp2tm1.1Sfis/CfreJ</td><td>The Jackson Labtorary&amp;nbsp;</td><td>026259</td><td>Foxp2&lt;sup&gt;fl/fl&lt;/sup&gt;</td></tr><tr><td>Dulbecco&amp;rsquo;s phosphate buffered saline</td><td>Corning&amp;nbsp;cellgro</td><td>21-030-CVR</td><td></td></tr></tbody></table>

stereotaxic surgery for genetic manipulation in striatal cells of neonatal mouse brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

For the first set of experiment, we microinjected 200 nL of AAV9.hSynapsin.HI.eGFP-Cre.WPRE.SV40 viruses (AAV-eGFP-Cre, 1/10 dilution in Dulbecco&#39;s phosphate buffered saline) that express the Cre DNA recombinase fused with GFP into P2 striatum of Ai14 mice. The Ai14 mice express tdTomato reporter gene upon Cre-mediated deletion of loxP-flanked (floxed) STOP cassette (Figure 2F). The brains were harvested at P14 for immunostaining of GFP and tdTomato. Many AAV transduced GFP-positive cells were present throughout the striatum (Figure 2A, C), indicating an extensive infection of striatal cells by AAV-eGFP-Cre viruses. Similar extensive expression of tdTomato was also found in the striatum (Figure 2B, C). Upon microscopic examination at high magnification, we found that nearly all GFP-positive striatal cells co-expressed the tdTomato reporter gene (Figure 2A&#39;-C&#39;). Moreover, tdTomato signals were detected in presumably axon terminals in the globus pallidus (Figure 2D) and the substantia nigra pars reticulata (Figure 2E), the target regions of striatonigral and striatopallidal projection neurons13. These results suggest a successful Cre-loxP mediated DNA recombination induced by AAV-mediated expression of Cre activity in neonatal striatal neurons.
For the second set of experiment, we microinjected 50 nL of AAV-eGFP-Cre viruses into the striatum of Foxp2fl/fl mice carrying loxP-flanked Foxp2 alleles at P2 (Figure 3A-C, F), and harvested the brain at P9. No Foxp2+;GFP+ double-labeled cells were found in the striatum, i.e., Foxp2 protein was absent in GFP-positive cells (Figure 3A&#39;-C&#39;). In control experiments, Foxp2+;GFP+ double-labeled cells were present in the striatum of Foxp2fl/fl mice with AAV-eGFP control viruses (Figure 3D) and in the striatum of heterozygous Foxp2fl/+ mice with AAV-eGFP-Cre viruses (Figure 3E). These results indicate that Foxp2 gene is specifically deleted in AAV-eGFP-Cre transduced cells of neonatal striatum.
Taken together the results of AAV-eGFP-Cre;Ai14 and AAV-eGFP-Cre;Foxp2fl/fl mice, the technique of stereotaxic neonatal brain surgery that we developed is amenable for conditionally delete genes in specific regions of neonatal mouse brains.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57270/57270fig1.jpg" /> 
Figure 1. Stereotaxic apparatus and homemade head-fixed holder for neonatal mice. (A) The stereotaxic injection system consists of the following devices: i. syringe pump (controller); ii. syringe pump (drive unit); iii. microliter syringe (10 &#181;L); iv. PE20 tubing adaptor; v. PE10 tube; vi. 30G injection needle; vii. stereotaxic apparatus; viii. a pipette tips box converted platform for neonatal pups; xi: crushed ice. (B) The height of the tissue embedding cassette is about 7 mm. The button of 1.5 mL centrifuge tube is about 15 mm long. (C) The mouse head is secured in the homemade head-fixed holder. The arrow indicates the landmark lambda in the head of neonatal mice. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57270/57270fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57270/57270fig2.jpg" /> 
Figure 2. Expression of tdTomato in the striatum of P14 Ai14 mice after AAV-eGFP-Cre mediated Cre-loxP DNA recombination. AAV-eGFP-Cre viruses were sterotaxically injected into P2 striatum of Ai14 mice. The brain was analyzed at P14. (A) Many GFP-positive cells are present throughout the striatum (Str). (B) Many tdTomato-positive cells are also present in the striatum. (C) The merged images show an extensive co-localization of GFP and tdTomato in the striatum. Confocal images of the boxed regions in A-C are shown at high magnification in A&#39;-C&#39;, respectively. All GFP-positive cells (green) co-express tdTomato (red) in the striatum. (D, E) The tdTomato signals are detected in the target regions of striatal projection neurons, including the globus pallidus (GP; D) and the substantia nigra pars reticulata (SNr; E). (F) Schematic drawings of AAV-eGFP-Cre and Ai14 transgenic constructs. Ctx: cortex; Sep: septum; Hipp: Hippocampus; MB: midbrain. Scale bar in A (for A-C), 200 &#181;m; A&#39; (for A&#39;-C&#39;), 10 &#181;m; D (for D and E), 100 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57270/57270fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57270/57270fig3.jpg" /> 
Figure 3. Conditional deletion of Foxp2 gene in the neonatal striatum by intrastriatal injecions of AAV-eGFP-Cre viruses. Intrastriatal injections of AAV-eGFP-Cre viruses were performed in P2 Foxp2fl/fl (A-C&#39;) and Foxp2fl/+ (E) mice. The brains were analyzed at P9. (A-C) GFP-positive cells (A, C, green) lack Foxp2 immunoreactivity (B, C, red) in the striatum of Foxp2fl/fl mice with AAV-eGFP-Cre viruses. The boxed regions in A-C are shown at high magnification in A&#39;-C&#39;, respectively. (D) Striatal cells co-express GFP and Foxp2 are present in the striatum that was injected with AAV-eGFP control viruses. (E) GFP-positive cells co-express Foxp2 (arrowheads) are present in the striatum of Foxp2fl/+ mice with AAV-eGFP-Cre viruses. (F) Schematic drawings of AAV-eGFP-Cre virus and Foxp2fl/fl transgenic mice. Scale bar in A (for A-C), 200 &#181;m; A&#39; (for A&#39;-C&#39;, D and E), 20 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57270/57270fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains at JoVE.com

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

stereotaxic-surgery-for-genetic-manipulation-striatal-cells-neonatal

Nanyang Technological University

Research

JoVE Journal

Neuroscience

14.9K Views.  National Yang-Ming University. We beschrijven een protocol van stereotaxic chirurgie met een zelfgemaakte hoofd-fixed device voor microinjecting reagentia in het striatum van neonatale muis hersenen. Deze techniek maakt het mogelijk specifieke genmanipulatie in neuronale cellen van bepaalde regio's van neonatale muis hersenen.

Video: Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

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 ...

Developmental Biology

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the function of various brain regions for behavior or other experimental outcomes is to implement a localized ablation of function. In humans, patient populations with localized brain lesions are often studied for deficits, in hopes of revealing the underlying function of the damaged area. In rodents, one can experimentally induce lesions of specific brain regions.
Lesion can be accomplished in several ways. Electrolytic lesions can cause localized damage but will damage a variety of cell types as well as traversing fibers from other brain regions that happen to be near the lesion site. Inducible genetic techniques using cell-type specific promoters may also enable site-specific targeting. These techniques are complex and not always practical depending on the target brain area. Excitotoxic lesion using stereotaxic surgery, by contrast, is one of the most reliable and practical methods of lesioning excitatory neurons without damaging local glial cells or traversing fibers.
Here, we present a protocol for stereotaxic infusion of the excitotoxin, N-methyl-D-aspartate (NMDA), into the basolateral amygdala complex. Using anatomical indications, we apply stereotaxic coordinates to determine the location of our target brain region and lower an injection needle in place just above the target. We then infuse our excitotoxin into the brain, resulting in excitotoxic death of nearby neurons. While our experimental subject of choice is a rat, the same methods can be applied to other mammals, with the appropriate adjustments in equipment and coordinates.
This method can be used on a variety of brain regions, including the basolateral amygdala1-6, other amygdala nuclei6, 7, hippocampus8, entorhinal cortex9 and prefrontal cortex10. It can also be used to infuse biological compounds such as viral vectors1, 11. The basic stereotaxic technique could also be adapted for implantation of more permanent osmotic pumps, allowing more prolonged exposure to a compound of interest.

Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

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 ...

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

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 ...

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous system. Overexpression of desired genes is just as possible as shRNA mediated loss-of-function studies. Therefore it offers a wide range of applications. The feasibility to target particular cells in a distinct area further increases the range of potential applications of this very useful method. For efficiently targeting specific regions knowledge about the subtleties, such as the embryonic stage, the voltage to apply and most importantly the position of the electrodes, is indispensable.
Here, we provide a detailed protocol that allows for specific and efficient in utero electroporation of several regions of the C57BL/6 mouse central nervous system. In particular it is shown how to transfect regions the develop into the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus. For this information about the appropriate embryonic stage, the appropriate voltage for the corresponding embryonic stage is provided. Most importantly an angle-map, which indicates the appropriate position of the positive pole, is depicted. This standardized protocol helps to facilitate efficient in utero electroporation, which might also lead to a reduced number of animals.

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

This protocol describes in detail how to specifically transfect different regions in the C57BL/6 central nervous system via in utero electroporation. Included in this protocol are detailed instructions for transfections of regions that develop into the cortex, hippocampus, thalamus, hypothalamus, lateral septal nucleus and striatum.

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous ...

Adaptable Angled Stereotactic Approach for Versatile Neuroscience Techniques

Stereotactic surgery is an essential tool in the modern neuroscience lab. However, the ability to precisely and accurately target difficult-to-reach brain regions still presents a challenge, particularly when targeting brain structures along the midline. These challenges include avoiding of the superior sagittal sinus and third ventricle and the ability to consistently target selective and discrete brain nuclei. In addition, more advanced neuroscience techniques (e.g., optogenetics, fiber photometry, and two-photon imaging) rely on targeted implantation of significant hardware to the brain, and spatial limitations are a common hindrance. Presented here is a modifiable protocol for stereotactic targeting of rodent brain structures using an angled coronal approach. It can be adapted to 1) mouse or rat models, 2) various neuroscience techniques, and 3) multiple brain regions. As a representative example, it includes the calculation of stereotactic coordinates for targeting of the mouse hypothalamic ventromedial nucleus (VMN) for an optogenetic inhibition experiment. This procedure begins with the bilateral microinjection of an adeno-associated virus (AAV) encoding a light-sensitive chloride channel (SwiChR++) to a Cre-dependent mouse model, followed by the angled bilateral implantation of fiberoptic cannulae. Using this approach, findings show that activation of a subset of VMN neurons is required for intact glucose counterregulatory responses to insulin-induced hypoglycemia.

Described here is a stereotactic procedure that can target challenging and difficult-to-reach brain regions (due to spatial limitations) using an angled coronal approach. This protocol is adaptable to both mouse and rat models and can be applied to diverse neuroscientific applications, including cannula implantation and microinjections of viral constructs.

Stereotactic surgery is an essential tool in the modern neuroscience lab. However, the ability to precisely and accurately target difficult-to-reach brain ...

Stereotaxic Intracranial Delivery of Chemicals, Proteins or Viral Vectors to Study Parkinson's Disease

Parkinson's disease (PD) is a progressive disorder traditionally defined by resting tremor and akinesia, primarily due to loss of dopaminergic neurons in the substantia nigra. Affected brain areas display intraneuronal fibrillar inclusions consisting mainly of alpha-synuclein (asyn) proteins. No animal model thus far has recapitulated all characteristics of this disease. Here, we describe the use of stereotaxic injection to deliver chemicals, proteins, or viral vectors intracranially in order to mimic various aspects of PD. These methods are well-established and widely used throughout the PD field. Stereotaxic injections are incredibly flexible; they can be adjusted in concentration, age of animal used for injection, brain area targeted and in animal species used. Combinations of substances allow for rapid variations to assess treatments or alter severity of the pathology or behavioral deficits. By injecting toxins into the brain, we can mimic inflammation and/or a severe loss of dopaminergic neurons resulting in substantial motor phenotypes. Viral vectors can be used to transduce cells to mimic genetic or mechanistic aspects. Preformed fibrillar asyn injections best recapitulate the progressive phenotype over an extended period of time. Once these methods are established, it can be economical to generate a new model compared to creating a new transgenic line. However, this method is labor intensive as it requires 30 minutes to four hours per animal depending on the model used. Each animal will have a slightly different targeting and therefore create a diverse cohort which on one hand can be challenging to interpret results from; on the other hand, help mimic a more realistic diversity found in patients. Mistargeted animals can be identified using behavioral or imaging readouts, or only after being sacrificed leading to smallercohort size after the study has already been concluded. Overall, this method is a rudimentary but effective way to assess a diverse set of PD aspects.

We describe how to successfully inject solutions into specific brain areas of rodents using a stereotaxic frame. This survival surgery is a well-established method used to mimic various aspects of Parkinson's disease.

Parkinson's disease (PD) is a progressive disorder traditionally defined by resting tremor and akinesia, primarily due to loss of dopaminergic neurons in ...

Rodent Stereotaxic Surgery

Stereotaxic (or stereotactic) surgery is a method used to manipulate the brain of living animals. This technique allows researchers to accurately target deep structures within the brain through the use of a stereotaxic atlas, which provides the 3D coordinates of each area with respect to anatomical landmarks on the skull. After the skull is exposed, anesthetized animals are mounted on a specialized instrument known as a stereotaxic frame, which enables the precise placement of experimental tools at the defined coordinates. Stereotaxic surgery is a versatile approach that can be used to generate lesions, manipulate gene expression, or deliver experimental agents to the brain.This video-article provides a general overview of the principles behind stereotaxic surgery, including instructions for using a stereotaxic atlas and the stereotaxic frame, and an introduction to reading the Vernier scale for measurement of probe movements. The subsequent discussion outlines the steps required to perform the surgical procedure. Lastly, a broad range of technical applications are presented, such as the insertion of electrical probes to measure brain activity and genetic manipulation of brain tissue.

Stereotaxic (or stereotactic) surgery is a method used to manipulate the brain of living animals. This technique allows researchers to accurately target ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

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