Preliminary Validation of Stereotaxic Injection Coordinates via Cryosectioning

Summary

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.

Abstract

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.

Introduction

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 interest^1,2,3, 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)....

Protocol

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.......

Discussion

This article has described a stable strategy to verify the accuracy of stereotaxic brain injections^5,6more 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.......

Materials

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