Alphavirus-Based Vectors - Viral Vector Approaches in Neurobiology and Brain Diseases - page 104

Pull the glass capillaries on an electrode puller to obtain micropi-
pettes with long tips, characteristic of sharp electrodes used for
intracellular recordings. Transfer the micropipettes into an auto-
clavable electrode holder. Ideally, foam holds the micropipettes so
that their tips remain intact. Autoclave the electrode holder with
the micropipettes.

3.12.1 Micropipette
Preparation

Mount the electrode holder onto the micromanipulator. Ideally,
the micromanipulator has both a coarse and a fi ne control. Fix the
micromanipulator onto a metal plate that contains a base for a
35-mm Petri dish. Place the setup according to biosafety level 2.
Use plastic tubing to connect the electrode holder via a three-way
valve to a 1-mL syringe. Insert a 35-mm plastic Petri dish into the
base on the metal plate. Add 2-3 mL cutting medium (prewarmed
to 37 °C) to the Petri dish. Focus the dissection microscope onto
the 35-mm Petri dish.

3.12.2 Assembly
of the Virus Injection Setup

Dilute the SFV stock by 10- to 1,000-fold in cutting medium.
In particular, when the GFP reporter gene is used, nondiluted
virus leads to extremely high infection rates and thus hinders the
identifi cation of individual GFP-positive cells. Back-fi ll an auto-
claved glass micropipette with 20-30

3.12.3 Micropipette
Loading

L of diluted SFV solution by
using a sterile Eppendorf microloader pipette tip. Insert the micro-
pipette into the airtight electrode holder. Sterilize small scissors
with 95 % ethanol and subsequent fl aming. Cut the micropipette
tip with the sterilized scissors to a fi nal diameter of approximately
20

μ

m. Verify that virus solution exits the micropipette tip when
positive pressure is applied from the 1-mL syringe.

μ

Transfer a cultured hippocampal slice into a 35-mm Petri dish by
using fl ame sterilized forceps. Lower the micropipette into the
pyramidal and/or granule cell layer by using the micromanipula-
tor. Inject virus with a short (<2 s) pressure pulse from the 1-mL
syringe. Release pressure via the three-way valve to stop the injec-
tion. Move the micropipette into a neighboring site and repeat
the injection. Choose 10-15 injection sites per slice and apply a
total of approximately 1

3.12.4 Virus Injection

L virus solution per slice. Bathe the
slice in roller-tube culture medium for 10-60 s to remove the
tetrodotoxin from the cutting medium as well as any nonbound
virus particles. This step thus lowers the biohazardous potential
of the slice. Return the slice to the culture system and incubate
for 1-3 days.

μ

SFV vectors can be used for direct stereotactic injections into rat
brain without any particular purifi cation or concentration proce-
dures [ 21 ]. However, if for some reason it is advantageous to pro-
vide the virus in a certain medium, any of the above-described
purifi cation procedures can be applied. Although no purifi cation is

3.13 In Vivo
Injections of SFV
Vectors