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 cage...

Optimizing Stereotaxic Microinjection Accuracy in Mice

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	<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>

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.

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

Preliminary Validation of Stereotaxic Injection Coordinates via Cryosectioning

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.

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, ...

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.

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