Viral Vector-Based Techniques for Optogenetic Modulation In Vivo - Viral Vector Approaches in Neurobiology and Brain Diseases - page 293

Allow cement to dry (5-10 min).

●

Slowly raise the cannula holder while pressing down on
the dried cement with tweezers.

●

The secured ferrule should keep in place, while the can-
nula holder is being raised.

●

20. Closing incision:

With tweezers, press skin together and apply a drop of
Vetbond™ tissue glue onto the subcutaneous tissue. Start
posterior and progress anteriorly along the incision
(Fig. 1 -cVI).

●

Alternatively, use surgical suture. Typically one suture is
done anterior to the implant and another one or two
posterior.

●

21. Remove mouse from stereotactic device and place in a separate
cage with fresh bedding and heated by a heat lamp.

If using isofl urane anesthesia: Before removing mouse,
remove ear bars, turn off isofl urane vaporizer, wait
1-2 min, turn off O 2 , then turn off active isofl urane clear-
ance system (if used).

●

Following long surgical procedures, it is recommended to
leave mouse with O 2 ventilation through the anesthesia
mask until detecting a toe pinch refl ex.

●

22. Wash syringe:

Withdraw a small volume of saline into the virus-contain-
ing syringe.

●

Eject virus and saline from syringe into biohazard vial. Do
not touch syringe tip to ethanol.

●

Fill and empty the syringe three times with DDW. Repeat
this step several times with clean DDW to assure effective
removal of virus from needle. Dispose of DDW according
to biohazard regulations.

●

Behavioral testing can be initiated 2-4 weeks following virus injec-
tion, depending on the type of virus and its titer. If light stimula-
tion is targeted at distal axons of the expressing cells, additional
time might be required for traffi cking of the opsin to the axons.
Histological examination can be performed to evaluate the time
required for expression and traffi cking of the optogenetic proteins
in the chosen paradigm. Here we briefl y describe a behavioral para-
digm used for evaluating the effects of optogenetic activation of
the primary mouse motor cortex following AAV-based expression
of ChR2(H134R)-mCherry in this region. The mouse used for

2.5 Optogenetic
Behavioral Testing