National Natural Science Foundation of China (grant NO. 82101249 to XY Sun), Postdoctoral Research Foundation of China (grant NO. 2022M722125 to XY Sun). Shanghai Sailing Program (grant NO. 21YF1425100 to SH Chen). Special Project for Clinical Research of Shanghai Municipal Health Commission (grant NO. 202340088 to J Zhou). National Natural Science Foundation of China (grant NO. 82101262 to X Zhang, grant NO. 82101287 to SH Chen).

All animal experiments were conducted in compliance with the Animal Research Reporting In Vivo Experiments (ARRIVE) guidelines and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The present study was approved by the Animal Care and Use Committee of Renji Hospital, Shanghai Jiaotong University School of Medicine. Eight-week-old C57BL/6J male mice were used for the present study. The animals were commercially obtained (see Table of Materials) and housed in standard cages (22 &#176;C &#177; 2 &#176;C, 12 h/12 h light/dark cycle, food, and water ad libitum).
1. Selection of target brain regions
NOTE: This is a sterile procedure. Ensure all surgical instruments are sterilized. Spray the gloves with alcohol prior to contact with surgical instruments. Avoid touching the tip of the instruments or the surgical area with gloves. Ensure the mouse is adequately anesthetized before starting the procedure by checking for the loss of righting reflexes and no response to cutaneous stimulation. Before the drilling and injection, closely monitor the respiratory rate (60-220 breaths per min, average 100-150 breaths per min) and the breathing amplitude (should be consistent and not excessively shallow or deep). Any significant changes in the respiratory rate or amplitude should be noted.
<ol>
	<li>Anesthetize the mouse with an intraperitoneal injection of 1.25% tribromoethanol (250 mg/kg) using a 29 G needle. Shave the hair on the skull once the righting reflex disappears. Secure the mouse in the stereotaxic frame (see Table of Materials) by fixing the upper incisors with a nose clamp and stabilizing the head using two ear bars.</li>
	<li>Apply ophthalmic ointment to prevent eye dryness. Disinfect the scalp by swabbing it three times with Anerdian (disinfectant solution; see Table of Materials). Inject 0.2 mL of 1% lidocaine subcutaneously using a 29 G insulin needle to provide local analgesia.</li>
	<li>Make a 0.5 cm incision using eye scissors to expose the cranium. Utilize a cotton swab dipped with 3% H2O2 to scrub the exposed skull to remove the periosteum. Allow the cranium to dry.</li>
	<li>Affix the drill to the stereotaxic instrument. Adjust the stereotaxic arms&#39; knobs to position the drill tip precisely at the bregma with the help of a magnifier and record the z-axis value using the LCD digital display.
	<ol>
		<li>Adjust the knobs and position the drill tip at the lambda and record the value of the z-axis. Adjust the nose clamp and ear bars to make two z-values closer, and repeat the steps above until the difference between the two z-values is less than 0.1 mm. 
		NOTE: The x, y, and z axes on the LCD digital display correspond to mediolateral (ML), anteroposterior (AP), and dorsoventral (DV), respectively. The bregma and lambda can be considered to be on the same horizontal plane once the difference between the two z-values is less than 0.1 mm.</li>
	</ol>
	</li>
	<li>Ensure the left-right levelness of the brain. Adjust the ear bars attached to the stereotaxic frame with the same scale. Take the laterodorsal tegmental nucleus, ventral part (LDTgV), for example; its stereotaxic coordinates are -5.2 for x, +0.8 for y, -4.0 for z, according to Paxinos and Franklin&#39;s mouse brain atlas16.
	<ol>
		<li>Adjust the ear bar to make the drill at (-5.2, +0.8) to measure the z value in the dorsal-ventral direction and move the drill to (-5.2, -0.8) to measure the z value. 
		NOTE: The left-right is considered to be at the equal level when the difference between the two z-values is less than 0.2 mm. The ear bars need to be inserted into the right place because the external auditory meatus is symmetric. If the difference between the two z-values is more than 0.5 mm, the ear bar may be inserted into the wrong place.</li>
	</ol>
	</li>
	<li>Position the drill to the bregma again and set three coordinates on the LCD digital display to zero. Position the drill above the region of interest using reference coordinates. Start the drill.
	<ol>
		<li>Gradually lower the drill using the vertical arm until it penetrates the cranium. Clear away debris and blood using cotton swabs. 
		NOTE: Be careful not to damage brain tissue.</li>
	</ol>
	</li>
</ol>
2. Preparing dye solution and brain microinjection
<ol>
	<li>Take 25 &#181;L of SDS-PAGE loading buffer containing bromophenol blue (see Table of Materials) into a 200 &#181;L microcentrifuge tube. Add 50 &#181;L of ddH2O to the tube to prepare the dye solution.</li>
	<li>Cover the cranial window with a saline-moistened cotton pad. Ensure microsyringe patency by aspirating and expelling saline repeatedly. Load 0.3 &#181;L of dye into the micro syringe. Attach the syringe and the motorized stereotaxic microinjector to the stereotaxic arm.</li>
	<li>Position the syringe tip at the bregma and set the coordinates to zero on the LCD digital display. Adjust the needle into the region of interest according to reference coordinates. Set the injection speed to 0.1 &#181;L/min and start the motorized microinjector, delivering 0.3 &#181;L of blue dye into the target region. Leave the syringe in position for at least 10 min before slowly withdrawing the injector. 
	NOTE: Different color dyes can be used in the same mice to distinguish closely located regions. Test other dyes for suitable concentrations based on their physical properties. It&#39;s recommended to use the bromophenol blue formula and thoroughly rinse the syringe before and after use to prevent blockages. Adjust injection volumes based on dye diffusion.</li>
</ol>
3. Brain tissue harvest and cryosectioning
<ol>
	<li>Release the mouse from the stereotaxic frame. Anesthetize the mouse with an intraperitoneal injection of 50 mg/kg pentobarbital sodium using a 29 G needle. Secure the mouse on a foam board with tape.</li>
	<li>Open both sides of the thorax to induce rapid suffocation. Make a 1-2 mm incision in the right atrial appendage. Insert an infusion needle into the left ventricle. Infuse 20 mL of pre-cooled saline and 20 mL of iced 4% paraformaldehyde solution.</li>
	<li>Decapitate the mouse with tissue dissecting scissors (following institutionally approved protocols). Remove the skin by making an incision along the midline from the neck to the nose and then expose the skull. Clear off residual muscle on the skull with forceps or scissors.</li>
	<li>Position the tip of one blade of the fine scissors between the foramen magnum and the brain, with the sharp edge facing the bone, at the rostral end of the mid-sagittal suture. Proceed to slide and cut the cranium along the mid-sagittal suture. Employ forceps to remove the skull, revealing the exposed brain. 
	NOTE: Exercise caution to avoid causing any damage to the brain during the operation. Before removing the bone chips, carefully peel off any meninges that may be attached to the skull. This precaution is crucial to prevent inadvertent slicing of the brain tissue during the process.</li>
	<li>Turn on the power of the rotary microtome cryostat (see Table of Materials) and set the chamber temperature to -20 &#176;C. Pour a little OCT compound to cover the surface of the specimen disc evenly. Place it into the cryo-chamber and allow the OCT to solidify.</li>
	<li>Attach the disc to the specimen head. Bring the blade closer to the block and trim the OCT block to make a flat plane that is parallel to the disc.</li>
	<li>Cut the brain in a coronal direction away from the injection site in a brain slice mold by a shaving blade. Place the brain tissue containing the injection site with the cut side down on the OCT flat plane.
	<ol>
		<li>Pour OCT onto the brain and put the disc into the cryo-chamber, allowing the specimen block to solidify. Pour OCT repeatedly onto the brain until the brain is fully embedded with OCT. 
		NOTE: Add the OCT compound layer by layer. Once the previous layer of OCT is frozen, pour the subsequent layer of OCT compound onto the brain.</li>
	</ol>
	</li>
	<li>Trim the specimen block until the target region is approaching. Make several sections from the injection site level. Collect the coronal brain slices into a 6-well plate containing room-temperature PBS using a small writing brush kept at -20 &#176;C. 
	NOTE: Set the cutting thickness to a larger one to trim the block, and then set the cutting thickness to 40 &#181;m as the blade approaches the injection site. If neural circuit tracers are used, the mice are not sacrificed immediately. Place the mouse in a sterile, postoperative recovery cage over a heating blanket for recovery from anesthesia. Return the mice to the original cage or a new cage once awake.</li>
</ol>
4. Imaging
<ol>
	<li>Wash the OCT with room temperature PBS. Use a brush to pick up brain sections and place them on a slide. Allow sections to air dry at room temperature.</li>
	<li>Observe the injected region visually or use a microscope when the target nuclei are minuscule. Compare the location of the injected dye with the corresponding region in the brain atlas. 
	NOTE: Adjust the values of the x, y, and z axes according to the direction and distance of the injection point deviation. It may be necessary to repeat all steps in this protocol multiple times to determine the coordinates accurately.</li>
</ol>

Optimizing Stereotaxic Microinjection Accuracy in Mice

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T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anterior+hypothalamic+parvalbumin+neurons+are+glutamatergic+and+promote+escape+behavior.">Anterior hypothalamic parvalbumin neurons are glutamatergic and promote escape behavior.</a> Curr Biol. 33 (15), 3215-3228 (2023).</li><li>Perens, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multimodal+3D+mouse+brain+atlas+framework+with+the+skull-derived+coordinate+system.">Multimodal 3D mouse brain atlas framework with the skull-derived coordinate system.</a> Neuroinformatics. 21 (2), 269-286 (2023).</li><li>Adhikari, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basomedial+amygdala+mediates+top-down+control+of+anxiety+and+fear.">Basomedial amygdala mediates top-down control of anxiety and fear.</a> Nature. 527 (7577), 179-185 (2015).</li><li>Tao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projections+from+infralimbic+cortex+to+paraventricular+thalamus+mediate+fear+extinction+retrieval.">Projections from infralimbic cortex to paraventricular thalamus mediate fear extinction retrieval.</a> Neurosci Bull. 37 (2), 229-241 (2021).</li><li>Haggerty, D. L., Grecco, G. G., Reeves, K. C., Atwood, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated+viral+vectors+in+neuroscience+research.">Adeno-associated viral vectors in neuroscience research.</a> Mol Ther Methods Clin Dev. 17, 69-82 (2020).</li><li>Ansarifar, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+volume+and+expression+time+in+an+aav-delivered+channelrhodopsin.">Impact of volume and expression time in an aav-delivered channelrhodopsin.</a> Mol Brain. 16 (1), 77 (2023).</li><li>Gonzalez, T. 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The authors declare no competing interests.

This article has described a stable strategy to verify the accuracy of stereotaxic brain injections5,6more quickly and simply before viral tracing, but the irreplaceable aspect of reporter gene expression in the brain region is crucial for brain region labeling. The blue dye we used allowed for the immediate visualization of the injection site.
Several critical steps in this protocol contribute to improving the accuracy of the injectio...

Nearly all modern neuromodulation tools, including in vivo calcium recording, optogenetic, and chemogenetic tools, require the use of stereotaxic coordinates to target the brain area of interest1,2,3, forming the foundation of neural manipulation. Stereotaxic coordinates for mouse brain regions are defined in relation to bregma and lambda, the bony landmarks on the cranium, forming the so-called skull-derived stereotaxic coordinate system. Either bregma or lambda can serve as the zero point of the three-dimensional coordinates. The three axes are anteroposterior (AP), mediolateral (ML), and dorsoventral (DV), representing the y, x, and z axes on the digital display of stereotaxic instruments. For well-known brain regions, the stereotaxic coordinate parameters of a specific area can be obtained from mouse brain atlases4 (e.g., Paxinos and Franklin&#39;s mouse brain in stereotaxic coordinates) and/or the published literature5,6. However, further validation is necessary due to variations in stereotaxic equipment and the age/populations of mice used by different researchers.
Structure is the basis of function. Neural circuits form the foundation for many brain functions, such as cognitive activities, emotion, memory, sensory, and motor functions1. Labeling the structure and manipulating the activity of neural circuits are vital for understanding the function of a specific neural circuit. Over the past decades, neural tracers have evolved through many generations; early research adopted wheat germ agglutinin (WGA) and phaseolus vulgaris agglutinin (PHA) as anterograde tracers, and fluorogold (FG), cholera toxin subunit B (CTB), carbocyanine as retrograde tracers. However, unlike viral tracers, these traditional neural tracers cannot integrate exogenous genes into the host, nor do they have cell type selectivity. Nowadays, the viral strategy has become an important proposition during basic neuroscience research. For different research purposes, various viral tools can be selected7,8. There are non-transsynaptic viruses, trans-multisynaptic viruses (retrograde and anterograde), and trans-monosynaptic viruses (retrograde and anterograde). Each category contains several types with respective characteristics.
The process of viral administration and expression is highly time- and resource-intensive, often taking weeks or even longer. Among various viral vectors, adeno-associated virus has been identified as a promising means for gene delivery, providing a wide window ranging from 3 to 8 weeks post-injection for the experimental procedure7,8. As AAV evolves, analysis can be performed 2-3 weeks after administration9,10. Other neural circuit tracers, such as pseudorabies virus (PRV) and rabies virus (RV), also require a tracing period of at least 2-7 days11,12,13,14,15. Thus, a preliminary verification of the injection site before observing fluorescence signals is both time- and cost-effective.
To facilitate a simple and rapid verification of stereotaxic injections, in this study, a dye is administered before viral vectors, and cryosectioning enables researchers to observe the injection site and track it within 30 min post-injection.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.0 &#181;L, Neuros Syringe, Model 7001 KH, 32 G, Point Style 3</td>
			<td>Hamilton</td>
			<td>65458-01</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#956;L pipette tips</td>
			<td>biosharp</td>
			<td>BS-200-T</td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL syringe</td>
			<td>Kindly group</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3%H2O2 solution</td>
			<td>Lircon Company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Shengyou Biotech</td>
			<td>20006</td>
			<td></td>
		</tr>
		<tr>
			<td>Anerdian</td>
			<td>Likang High-tech</td>
			<td>31001002</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti roll plate</td>
			<td>Leica</td>
			<td>14047742497</td>
			<td></td>
		</tr>
		<tr>
			<td>BD insulin syringe</td>
			<td>Becton,Dickinson and Company</td>
			<td>328421</td>
			<td></td>
		</tr>
		<tr>
			<td>Bend toothed dissecting forceps</td>
			<td>Jinzhong</td>
			<td>JD1050</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellsens dimension software</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton swab</td>
			<td>Fisher Scientific</td>
			<td>23-400-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Dapi-Fluoromount-G</td>
			<td>Southernbiotech</td>
			<td>0100-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Drill</td>
			<td>Longxiang</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine brushes</td>
			<td>HWAHONG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine scissors</td>
			<td>Jinzhong</td>
			<td>y00030</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent microscopy</td>
			<td>Olympus</td>
			<td>BX63</td>
			<td></td>
		</tr>
		<tr>
			<td>freezing microtome</td>
			<td>Leica</td>
			<td>CM1950</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemostatic forceps straight with tooth</td>
			<td>Jinzhong</td>
			<td>J31010</td>
			<td></td>
		</tr>
		<tr>
			<td>Infusion needle 0.7 mm</td>
			<td>Kindly group</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lidocaine hydrochloride injection</td>
			<td>Harvest Pharmaceutical Company</td>
			<td>71230803</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnifying glass</td>
			<td>M&#38;G Chenguang Stationery</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Male C57/BL mice</td>
			<td>The Shanghai Institute of Planned Parenthood Research&#8211;BK Laboratory</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mice coronal brain slice mold</td>
			<td>RWD Life Science</td>
			<td>68713</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tube</td>
			<td>biosharp</td>
			<td>BS-02-P</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome blades</td>
			<td>Leica</td>
			<td>819</td>
			<td></td>
		</tr>
		<tr>
			<td>Ophthalmic ointment</td>
			<td>Cisen Pharmaceutical Company</td>
			<td>G23HDM9M4S5</td>
			<td></td>
		</tr>
		<tr>
			<td>paraformaldehyde</td>
			<td>Biosharp</td>
			<td>BL539A</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pumps</td>
			<td>Harvard Apparatus</td>
			<td>70-4507</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Servicebio</td>
			<td>G4202</td>
			<td></td>
		</tr>
		<tr>
			<td>Piette 2-200 &#956;L</td>
			<td>thermofisher</td>
			<td>4642080</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE sample loading containing bromophenol blue</td>
			<td>Beyotime</td>
			<td>P0015A</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaving blades</td>
			<td>BFYING</td>
			<td>91560618</td>
			<td></td>
		</tr>
		<tr>
			<td>Slides</td>
			<td>Citotest Scientific</td>
			<td>188105</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotaxic apparatus</td>
			<td>RWD Life Science</td>
			<td>68807</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight toothed dissecting forceps</td>
			<td>Jinzhong</td>
			<td>JD1060</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Holder</td>
			<td>RWD Life Science</td>
			<td>68206</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue scissors</td>
			<td>Jinzhong</td>
			<td>J21040</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek O.C.T compound</td>
			<td>Sakura</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Tribromoethanol</td>
			<td>Aibei Biotechnology</td>
			<td>M2910</td>
			<td></td>
		</tr>
	</tbody>
</table>

preliminary validation of stereotaxic injection coordinates via cryosectioning

Stereotaxic injection of a specific brain region constitutes a fundamental experimental technique in basic neuroscience. Researchers commonly base their choice of stereotaxic injection parameters on mouse brain atlases or published materials that employed various populations/ages of mice and different stereotaxic equipment, necessitating further validation of the stereotaxic coordinate parameters. The efficacy of calcium imaging, chemogenetic, and optogenetic manipulations relies on the precise expression of reporter genes within the region of interest, often requiring several weeks of effort. Thus, it is a time-consuming task if the coordinates of the target brain region are not verified in advance. Using an appropriate dye instead of a virus and implementing cryosectioning, researchers can observe the injection site immediately following dye administration. This facilitates timely adjustments to coordinate parameters in cases where discrepancies exist between the actual injection site and the theoretical position. Such adjustments significantly enhance the accuracy of viral expression within the target region in subsequent experiments.

This study successfully identified the injection site within 30 min using the demonstrated method. Initially, an SDS-PAGE sample loading solution containing bromophenol blue was injected into the LDTgV in the C57/BL mice. Figure 1A shows a schematic representation of the dye solution injection. The distribution of the blue dye in the LDTgV is illustrated in Figure 1B.
Bromophenol blue was also injected into the mPFC prelimbic cortex, ...

Author Spotlight: Accelerating Cognitive Impairment Research in Mice Through Stereotaxic Injection and Cost-Effective Dyes

The present protocol describes a practical strategy to expedite the verification step of stereotaxic injection coordinates before conducting viral tracing using dyes and frozen sections.

Preliminary Validation of Stereotaxic Injection Coordinates via Cryosectioning

Stereotaxic injection of a specific brain region constitutes a fundamental experimental technique in basic neuroscience. Researchers commonly base their ...

Our research primarily focus on investigating the neural circuits mechanism underlying cognitive impairment in mice. And we frequently employ brain stereotaxic techniques in these studies. Our objective is to optimize and accelerate the verification process for stereotaxic injection sites using dyes, ensuring that it becomes a simple and rapid procedure.Various strategies have become crucial in neuroscience research through stereotaxic injection of virus. This is followed by observation of fluorescent markers in tissue slice to confirm the injection sites, a process that takes several weeks to complete. Our protocol offers advantages over other techniques by saving at least three days of dehydration time and using a cost-effective dye for quicker identification of injection sites.It may save weeks compared with no preliminary verification.

preliminary-validation-stereotaxic-injection-coordinates-via

Research

JoVE Journal

Neuroscience

927 Views.  Shanghai Jiaotong University, School of Medicine. Our research primarily focus on investigating the neural circuits mechanism underlying cognitive impairment in mice. And we frequently employ brain stereotaxic techniques in these studies. Our objective is to optimize and accelerate the verification process for stereotaxic injection sites using dyes, ensuring that it becomes a simple and rapid procedure.Various strategies have become crucial in neuroscience research through stereotaxic injection of virus. This is followed by observatio...

Video: Author Spotlight: Accelerating Cognitive Impairment Research in Mice Through Stereotaxic Injection and Cost-Effective Dyes

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 ...

Building An Open-source Robotic Stereotaxic Instrument

This protocol includes the designs and software necessary to upgrade an existing stereotaxic instrument to a robotic (CNC) stereotaxic instrument for around $1,000 (excluding a drill), using industry standard stepper motors and CNC controlling software. Each axis has variable speed control and may be operated simultaneously or independently. The robot&#39;s flexibility and open coding system (g-code) make it capable of performing custom tasks that are not supported by commercial systems. Its applications include, but are not limited to, drilling holes, sharp edge craniotomies, skull thinning, and lowering electrodes or cannula. In order to expedite the writing of g-coding for simple surgeries, we have developed custom scripts that allow individuals to design a surgery with no knowledge of programming. However, for users to get the most out of the motorized stereotax, it would be beneficial to be knowledgeable in mathematical programming and G-Coding (simple programming for CNC machining).
The recommended drill speed is greater than 40,000 rpm. The stepper motor resolution is 1.8&#176;/Step, geared to 0.346&#176;/Step. A standard stereotax has a resolution of 2.88 &#956;m/step. The maximum recommended cutting speed is 500 &#956;m/sec. The maximum recommended jogging speed is 3,500 &#956;m/sec. The maximum recommended drill bit size is HP 2.

This protocol includes the designs and software necessary to upgrade an existing stereotaxic instrument to a robotic (computer numeric controlled; CNC) stereotaxic instrument for around $1,000 (excluding a drill).

This protocol includes the designs and software necessary to upgrade an existing stereotaxic instrument to a robotic (CNC) stereotaxic instrument for ...

Designing, Packaging, and Delivery of High Titer CRISPR Retro and Lentiviruses via Stereotaxic Injection

Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique has been widely used to encode fluorescent proteins, opto- or chemo-genetic controllers of cell activity, or heterologous expression of human genes in model organisms. These viruses have also successfully been used to deliver recombinases to relevant target sites in transgenic animals, or even deliver small hairpin or micro RNAs in order to manipulate gene expression. While these techniques have been fruitful, they rely on transgenic animals (recombinases) or frequently lack high efficacy and specificity (shRNA/miRNA). In contrast, the CRISPR/Cas system uses an exogenous Cas nuclease which targets specific sites in an organism's genome via an exogenous guide RNA in order to induce double stranded breaks in DNA. These breaks are then repaired by non-homologous end joining (NHEJ), producing insertion and deletion (indel) mutations that can result in deleterious missense or nonsense mutations. This manuscript provides detailed methods for the design, production, injection, and validation of single lenti/retro virus particles that can stably transduce neurons to express a fluorescent reporter, Cas9, and sgRNAs to knockout genes in a model organism.

The CRISPR/Cas9 system offers the potential to make targeted genome editing accessible and affordable to the scientific community. This protocol is intended to demonstrate how to create viruses that will knockout a gene of interest using the CRISPR/Cas9 system, and then inject them stereotaxically into the adult mouse brain.

Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique ...

Simultaneous Cryosectioning of Multiple Rodent Brains

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In neuroscience, as well as a plethora of other scientific fields, these techniques are used. Immunohistochemistry can be done on slide mounted tissue or free-floating sections. Preparing slide-mounted samples is a time intensive process. The following protocol for a technique, called the Megabrain, reduced the time taken to cryosection and mount brain tissue by up to 90% by combining multiple brains into a single frozen block. Furthermore, this technique reduced variability seen between staining rounds, in a large histochemical study. The current technique has been optimized for using rodent brain tissue in downstream immunohistochemical analyses; however, it can be applied to different scientific fields that use cryosectioning.

Here, we present a protocol to freeze and section brain tissue from multiple animals as a timesaving alternative to processing single brains. This reduces staining variability during immunohistochemistry and reduces time cryosectioning and imaging.

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In ...

Induction of Leptomeningeal Cells Modification Via Intracisternal Injection

The protocol outlined here describes how to safely and manually inject solutions through the cisterna magna while eliminating the risk of damage to the underlying parenchyma. Previously published protocols recommend using straight needles that should be lowered to a maximum of 1-2 mm from the dural surface. The sudden drop in resistance once the dural membrane has been punctured makes it difficult to maintain the needle in a steady position. Our method, instead, employs a needle bent at the tip that can be stabilized against the occipital bone of the skull, thus preventing the syringe from penetrating into the tissue after perforation of the dural membrane. The procedure is straightforward, reproducible, and does not cause long-lasting discomfort in the operated animals. We describe the intracisternal injection strategy in the context of genetic fate mapping of vascular leptomeningeal cells. The same technique can, furthermore, be utilized to address a wide range of research questions, such as probing the role of leptomeninges in neurodevelopment and the spreading of bacterial meningitis, through genetic ablation of genes putatively implicated in these phenomena. Additionally, the procedure can be combined with an automatized infusion system for a constant delivery and used for tracking cerebrospinal fluid movement via injection of fluorescently labelled molecules.

We describe an intracisternal injection that employs a needle bent at the tip that can be stabilized to the skull, thus eliminating the risk of damage to the underlying parenchyma. The approach can be used for genetic fate mapping and manipulations of leptomeningeal cells and for tracking cerebrospinal fluid movement.

The protocol outlined here describes how to safely and manually inject solutions through the cisterna magna while eliminating the risk of damage to the ...

Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging

A miniature fluorescence microscope (miniscope) is a potent tool for in vivo calcium imaging from freely behaving animals. It offers several advantages over conventional multi-photon calcium imaging systems: (1) compact; (2) light-weighted; (3) affordable; and (4) allows recording from freely behaving animals. This protocol describes brain surgeries for deep brain in vivo calcium imaging using a custom-developed miniscope recording system. The preparation procedure consists of three steps, including (1) stereotaxically injecting the virus at the desired brain region of a mouse brain to label a specific subgroup of neurons with genetically encoded calcium sensor; (2) implantation of gradient-index (GRIN) lens that can relay calcium image from deep brain region to the miniscope system; and (3) affixing the miniscope holder over the mouse skull where miniscope can be attached later. To perform in vivo calcium imaging, the miniscope is fastened onto the holder, and neuronal calcium images are collected along with simultaneous behavior recordings. The present surgery protocol is compatible with any commercial or custom-built single-photon and two-photon imaging systems for deep brain in vivo calcium imaging.

Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging

Miniscope in vivo calcium imaging is a powerful technique to study neuronal dynamics and microcircuits in freely behaving mice. This protocol describes performing brain surgeries to achieve good in vivo calcium imaging using a miniscope.

A miniature fluorescence microscope (miniscope) is a potent tool for in vivo calcium imaging from freely behaving animals. It offers several advantages ...

Stereotaxic Surgical Approach to Microinject the Caudal Brainstem and Upper Cervical Spinal Cord via the Cisterna Magna in Mice

Stereotaxic surgery to target brain sites in mice is commonly guided by skull landmarks. Access is then obtained via burr holes drilled through the skull. This standard approach can be challenging for targets in the caudal brainstem and upper cervical cord due to specific anatomical challenges as these sites are remote from skull landmarks, leading to imprecision. Here we outline an alternative stereotaxic approach via the cisterna magna that has been used to target discrete regions of interest in the caudal brainstem and upper cervical cord. The cisterna magna extends from the occipital bone to the atlas (i.e., the second vertebral bone), is filled with cerebrospinal fluid, and is covered by dura mater. This approach provides a reproducible route of access to select central nervous system (CNS) structures that are otherwise hard to reach due to anatomical barriers. Furthermore, it allows for direct visualization of brainstem landmarks in close proximity to the target sites, increasing accuracy when delivering small injection volumes to restricted regions of interest in the caudal brainstem and upper cervical cord. Finally, this approach provides an opportunity to avoid the cerebellum, which can be important for motor and sensorimotor studies.

Stereotaxic Surgical Approach to Microinject the Caudal Brainstem and Upper Cervical Spinal Cord via the Cisterna Magna in Mice

Stereotaxic surgery to target brain sites in mice commonly involves access through the skull bones and is guided by skull landmarks. Here we outline an alternative stereotaxic approach to target the caudal brainstem and upper cervical spinal cord via the cisterna magna that relies on direct visualization of brainstem landmarks.

Stereotaxic surgery to target brain sites in mice is commonly guided by skull landmarks. Access is then obtained via burr holes drilled through the skull. ...

Mouse Dorsal Root Ganglion Cryosectioning

A Procedure for Mouse Dorsal Root Ganglion Cryosectioning

High-quality mouse dorsal root ganglion (DRG) cryostat sections are crucial for proper immunochemistry staining and RNAscope studies in the research of inflammatory and neuropathic pain, itch, as well as other peripheral neurological conditions. However, it remains a challenge to consistently obtain high-quality, intact, and flat cryostat sections onto glass slides because of the tiny sample size of the DRG tissue. So far, there is no article describing an optimal protocol for DRG cryosectioning. This protocol presents a step-by-step method to resolve the frequently encountered difficulties associated with DRG cryosectioning. The presented article explains how to remove the surrounding liquid from the DRG tissue samples, place the DRG sections on the slide facing the same orientation, and flatten the sections on the glass slide without curving up. Although this protocol has been developed for cryosectioning the DRG samples, it can be applied for the cryosectioning of many other tissues with a small sample size.

Author Spotlight: A Procedure for Mouse Dorsal Root Ganglion Cryosectioning  

Presented here is the development for consistently acquiring high-quality dorsal root ganglion cryostat sections.

High-quality mouse dorsal root ganglion (DRG) cryostat sections are crucial for proper immunochemistry staining and RNAscope studies in the research of ...