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

<ol>
	<li>Liu, D., 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=Brain-derived+neurotrophic+factor-mediated+projection-specific+regulation+of+depressive-like+and+nociceptive+behaviors+in+the+mesolimbic+reward+circuitry.">Brain-derived neurotrophic factor-mediated projection-specific regulation of depressive-like and nociceptive behaviors in the mesolimbic reward circuitry.</a> Pain. 159 (1), 175 (2018).</li><li>Gan, Z., 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=Layer-specific+pain+relief+pathways+originating+from+primary+motor+cortex.">Layer-specific pain relief pathways originating from primary motor cortex.</a> Science. 378 (6626), 1336-1343 (2022).</li><li>Laing, B. 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. 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=Structure-guided+AAV+capsid+evolution+strategies+for+enhanced+CNS+gene+delivery.">Structure-guided AAV capsid evolution strategies for enhanced CNS gene delivery.</a> Nat Protoc. 18 (11), 3413-3459 (2023).</li><li>Sun, X. 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=Two+parallel+medial+prefrontal+cortex-amygdala+pathways+mediate+memory+deficits+via+glutamatergic+projection+in+surgery+mice.">Two parallel medial prefrontal cortex-amygdala pathways mediate memory deficits via glutamatergic projection in surgery mice.</a> Cell Rep. 42 (7), 112719 (2023).</li><li>Koren, 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=Insular+cortex+neurons+encode+and+retrieve+specific+immune+responses.">Insular cortex neurons encode and retrieve specific immune responses.</a> Cell. 184 (25), 6211 (2021).</li><li>Poller, W. C., 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=Brain+motor+and+fear+circuits+regulate+leukocytes+during+acute+stress.">Brain motor and fear circuits regulate leukocytes during acute stress.</a> Nature. 607 (7919), 578-584 (2022).</li><li>Huang, L., 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=A+visual+circuit+related+to+habenula+underlies+the+antidepressive+effects+of+light+therapy.">A visual circuit related to habenula underlies the antidepressive effects of light therapy.</a> Neuron. 102 (1), 128-142 (2019).</li><li>Hu, Z., 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=A+visual+circuit+related+to+the+periaqueductal+gray+area+for+the+antinociceptive+effects+of+bright+light+treatment.">A visual circuit related to the periaqueductal gray area for the antinociceptive effects of bright light treatment.</a> Neuron. 110 (10), 1712-1727 (2022).</li><li>Du, W., 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=Directed+stepwise+tracing+of+polysynaptic+neuronal+circuits+with+replication-deficient+pseudorabies+virus.">Directed stepwise tracing of polysynaptic neuronal circuits with replication-deficient pseudorabies virus.</a> Cell Rep Methods. 3 (6), 100506 (2023).</li><li>Paxinos, G., Franklin, K. B. J., Franklin, K. B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The mouse brain in stereotaxic coordinates. 2nd ed. , (2001).</li></ol>

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 ...

preliminary-validation-stereotaxic-injection-coordinates-via

Research

JoVE Journal

Neuroscience

1.4K Views.  Shanghai Jiaotong University, School of Medicine. 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.

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

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 ...

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 ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

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 ...

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to the interphotoreceptor/subretinal compartment that has direct exposure to photoreceptors and the retinal pigment epithelium (RPE). Subretinal injections of plasminogen as well as recent preclinical and clinical trials have demonstrated safety and/or efficacy of delivering viral vectors and stem cells to individuals with advanced retinal disease. Mouse models of retinal disease, particularly hereditary retinal dystrophies, are essential for testing these therapies. The most common injection procedure in rodents is to use small transcorneal or transcleral incisions with an anterior approach to the retina. With this approach, the injection needle penetrates the neurosensory retina disrupting the underlying RPE and on insertion can easily nick the lens, causing lens opacification and impairment of noninvasive imaging. Accessing the subretinal space via a transcleral, posterior approach avoids these problems: the needle crosses the sclera approximately 0.5 mm from the optic nerve, without retinal penetration and avoids disrupting the vitreous. Collateral damage is limited to that associated with the focal sclerotomy and the effects of a transient, serous retinal detachment. The simplicity of the method minimizes ocular injury, ensures rapid retinal reattachment and recovery, and has a low failure rate. The minimal damage to the retina and RPE allows for clear assessment of the efficacy and direct effects of the therapeutic agents themselves. This manuscript describes a novel subretinal injection technique that can be used to target viral vectors, pharmacological agents, stem cells or induced pluripotent stem (iPS) cells to the subretinal space in mice with high efficacy, minimal damage, and fast recovery.

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections are the most common technique for delivering large therapeutic agents such as proteins and viral vectors to photoreceptors and the retinal pigment epithelium. An alternative method in mice that successfully targets the subretinal space with minimal collateral damage and fast recovery times is described here.

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to ...

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.

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, ...

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 ...

Stereotaxic Surgery for Implantation of Microelectrode Arrays in the Common Marmoset (Callithrix jacchus)

Marmosets (Callithrix jacchus) are small non-human primates that are gaining popularity in biomedical and preclinical research, including the neurosciences. Phylogenetically, these animals are much closer to humans than rodents. They also display complex behaviors, including a wide range of vocalizations and social interactions. Here, an effective stereotaxic neurosurgical procedure for implantation of recording electrode arrays in the common marmoset is described. This protocol also details the pre- and postoperative steps of animal care that are required to successfully perform such a surgery. Finally, this protocol shows an example of local field potential and spike activity recordings in a freely behaving marmoset 1 week after the surgical procedure. Overall, this method provides an opportunity to study the brain function in awake and freely behaving marmosets. The same protocol can be readily used by researchers working with other small primates. In addition, it can be easily modified to allow other studies requiring implants, such as stimulating electrodes, microinjections, implantation of optrodes or guide cannulas, or ablation of discrete tissue regions.

Stereotaxic Surgery for Implantation of Microelectrode Arrays in the Common Marmoset Callithrix jacchus

This work presents a protocol to perform a stereotaxic, neurosurgical implantation of&#160;microelectrode arrays in the common marmoset. This method specifically enables electrophysiological recordings in freely behaving animals but can be easily adapted to any other similar neurosurgical intervention in this species (e.g., cannula for drug administration or electrodes for brain stimulation).

Preliminary Validation of Stereotaxic Injection Coordinates via Cryosectioning

Summary

Explore More Videos

Chapters in this video

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

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

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

Building An Open-source Robotic Stereotaxic Instrument

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

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

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

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

Simultaneous Cryosectioning of Multiple Rodent Brains

Stereotaxic Surgery for Implantation of Microelectrode Arrays in the Common Marmoset (Callithrix jacchus)