Viral Vectors for Optogenetics of Hypothalamic Neuropeptides - Viral Vector Approaches in Neurobiology and Brain Diseases - page 306

CAG-LSL-opsin; JAX mice stock number: 012567 and 012569,
[ 17 ]). It has to be kept in mind that expression displays specifi city
only at certain points in space and time. However, in particular
transient Cre expression at any developmental state would lead to
irreversible recombination and permanent opsin-expression.
Moreover, neurons, molecularly defi ned as a specifi c cell type, might
serve fundamental different functions by virtue of differential wir-
ing [ 1 , 32 , 55 ] and can be separately examined with distinct virus
delivery only.
Noteworthy are fi nally several, to some extent inducible,
approaches for opsin expression, including a tamoxifen-inducible
Cre line crossbred to Cre-dependent opsin transgenes [ 62 ], mice
with bidirectional opsin expression (ChR2 and NpHR) under tet-
racycline transactivator (tTA)-control [ 63 ] and the “Virus-
mediated Genetic Activity-Induced Tagging” (vGAIT)-technique,
serving for opsin-tagging to previously activated neurons [ 64 ].

For the optogenetic experiments described here we used Wistar
rats, but it is very likely that other strains can also be effi ciently
used. We injected rAAVs 8-10 weeks after birth and waited a mini-
mum of 1 month to allow for suffi cient virus expression.

2.3 In Vitro
Optogenetics
(See Fig. 1 )

2.3.1 Animals

Coupling electrophysiological recordings to optical stimulation
requests a combination of basic, but important techniques, which
are listed below. All of the different equipment is of crucial impor-
tance for the experiments and should be carefully checked before
starting a project.

2.3.2 Material

General Considerations

1. A complete patch-clamp setup that includes:
2. A recording chamber that can be rapidly perfused with artifi -
cial cerebrospinal fl uid (ACSF).
3. Differential interference contrast (DIC) or oblique light com-
patible with fl uorescent imaging and a background light inten-
sity as small as possible to limit possible unwanted
channelrhodopsin stimulation.
4. Fluorescence microscope to visualize the infected cells through
the fl uorescent reporter-protein fused with the channelrho-
dopsin. A mercury lamp of minimal 50 W (short Arc 103 W/2,
OSRAM, Augsburg, Germany; ~5 mW/mm 2 ), coupled with
appropriate fi lter cube and a shutter (VS25S22M1R1,
Uniblitz, San Francisco, USA) allows a very effi cient and easy
optical stimulation. Another user-friendly and more low-cost
way to deliver precise light pulses is the use of LEDs (Luxeon ®
Star LEDs, Quandica Developments Inc., Ontario, Canada),
which can be easily triggered at the required frequencies. Final
light intensity will be set between 1 and 5 mW/mm 2 . This
intensity should be similar to the optical stimulation that is
used with a blue laser coupled in a similar manner.

Detailed Material