Applying Stereotactic Injection Technique to Study Genetic Effects on Animal Behaviors

Summary

Stereotactic injection of lentiviruses expressing cDNAs or shRNAs can modulate gene expression in specific brain areas of mice. Here, we present a protocol to combine stereotactic injections with behavioral tasks, such as the Open Field Test (OFT) and the Forced Swim Test (FST).

Abstract

Stereotactic injection is a useful technique to deliver high titer lentiviruses to targeted brain areas in mice. Lentiviruses can either overexpress or knockdown gene expression in a relatively focused region without significant damage to the brain tissue. After recovery, the injected mouse can be tested on various behavioral tasks such as the Open Field Test (OFT) and the Forced Swim Test (FST). The OFT is designed to assess locomotion and the anxious phenotype in mice by measuring the amount of time that a mouse spends in the center of a novel open field. A more anxious mouse will spend significantly less time in the center of the novel field compared to controls. The FST assesses the anti-depressive phenotype by quantifying the amount of time that mice spend immobile when placed into a bucket of water. A mouse with an anti-depressive phenotype will spend significantly less time immobile compared to control animals. The goal of this protocol is to use the stereotactic injection of a lentivirus in conjunction with behavioral tests to assess how genetic factors modulate animal behaviors.

Introduction

The mouse has been widely used in neurobiology because it is easy to manipulate genetically. Gene knockout techniques allow researchers to investigate how each genetic factor shapes mouse behaviors. Moreover, the cre-loxp system provides a valuable tool for tissue- and cell type- specific gene knockout in mice, which enables researchers to study gene function in different tissues.¹ However, in practice, expression patterns of cre promoters are difficult to control, and so far many established cre drivers have not achieved region-specific expression.^2,3

Alternatively, stereotactic injection is a method that targets spec....

Protocol

NOTE: All protocols involving animals were followed in accordance with the animal care guidelines of The Pennsylvania State University, IACUC #44057

1. Lentivirus Production

NOTE: The day before transfection, LentiX-293 cells should be at 80% confluency.

1. Rinse cells with DMEM just before transfection and incubate in 10 ml of DMEM/10% FBS with Penicillin (100 IU/ml) and Streptomycin (100 µg/ml) for 5 min at RT.
2. Dilute DNA and Polyethylenimine (PEI) (1 mg/ml) in 1:3 ratio (1 µg of DNA: 3 µl of PEI) in 1 ml of DMEM and vortex for 1 min. The volume of DNA added is dependent o....

Results

Accurate stereotactic injection relies heavily on setting the correct coordinates. The tip of the needle used to inject the virus should be set directly on the intersection of bregma and the interaural line (Figure 1). It is helpful to use a stereomicroscope to ascertain whether or not the needle is placed correctly. When looking through the microscope, the needle should be positioned so that if the virus were injected, it would land directly on the intersection of bregma and the interaural line. That is.......

Discussion

Successful stereotactic injections rely on three factors: keeping the mouse alive, setting the correct zero point for coordinates (tip of the needle on the intersection of bregma and the interaural line), and setting the right depth to the zero point (tip of the needle just touching the exterior of the brain tissue). The viability of mice is important. Surgery survival can be aided by making sure that the mouse is properly anesthetized and receives adequate analgesic. Pain is known to be a major cause of poor recovery af.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Stereotactic Apparatus item 51725	Stoelting co.	51725
Quintessential stereotaxic pump	Stoelting co.	53311
Injection Styringe, 65 RN	Hamilton	7633-01
DMEM	Sigma-Aldrich	D5796
PEI	Polysciences Inc.	23966
Scissors	Fine Surgical Tools	14084-08
Blunt end forceps	Fine Surgical Tools	11002-12
Needle holder	Fine Surgical Tools	12001-13
Drill	Ram Products Inc	Microtorque control box, Tech2000 handpiece	with pedal
Glass bead sterilizer	Inotech	IS-400
Absorbable sutures	Unify	M-K518r19	Absorbable, reverse cutting
Cotton swabs	VWR	89031-270
Heating pad	Gaymar	T-pump TP-500 PN11184-000
Artificial tears	Rugby	NDC 0536-6550-91
Disposable syringe	BD Syringe	309623
Cloth
Gloves	Ansell	Senseitouch #7823
Avertin or other anesthetic	see recipe citation
Ketoprofen or other analgesic	see veternarian
Tracking software	Noldus	Ethovision XT
Tracking Camera	Noldus	Media Recorder