Viral Vectors for Optogenetics of Hypothalamic Neuropeptides - Viral Vector Approaches in Neurobiology and Brain Diseases - page 312

3. Free the skull by removing all skin and conjunctive tissue from
the skull. Use oxygenated water (3 %) to clean it and ensure
there is no more tissue or blood.
4. Drill holes at the precise stereotaxic coordinates above the
region of interest. Insert two small screws into the skull on
both sides of the holes to create attachment points for a dental
cement cap. Gently lower the cannulae down into the holes.
5. Secure the placement of the cannulae by pouring dental
cement on the skull of the rat and place the dummy caps into
the cannulae.

1. Optical fi bers should be gently inserted in the guide cannulae.
Anesthesia of the animal is not a prerequisite if it has been cor-
rectly and suffi ciently handled and habituated beforehand for
at least 1 week.
2. Length of the optical fi ber should be precisely defi ned i.e. (a)
long enough to let the animal move freely and (b) short
enough to avoid the rat biting the cable.
3. Light stimulation must be based on what has been found to
work effi ciently in vitro and on the scientifi c question to be
addressed. In our case, the goal was to compare previous exog-
enous application of oxytocin with evoked release of endoge-
nous oxytocin. We therefore used a frequency of 30 Hz and
pulse duration of 10 ms, in order to induce a maximum num-
ber of action potentials for highest release of OT over a short
period of time.

2.4.3 Stimulation
with Blue Light

4. After the experiment, rapidly remove the optical fi ber. Gently
handle the rat—this point is particularly critical—in order to
avoid breaking the optical fi ber. A fi ber that remains behind in
the cannula precludes further experimentations with the ani-
mal and can, moreover, potentially cause cerebral damage.

3 Important General Notes on Optogenetic Regulation of Neuropeptide Release

Using optogenetics, several concerns should be taking into account
that we have listed below.
One should consider the potential of backpropagation of axo-
nal action potentials (AP), mainly when using in vivo optogenetics
[ 14 ]. Indeed, De Kock et al. [ 67 ] showed, using capacitance mea-
surements, that a single antidromic AP is suffi cient to elicit exocy-
tosis of OT from large-density core vesicles (LDCVs) in dendrites.
On the contrary, Ludwig et al. [ 68 ] showed that antidromic activa-
tion of SON neurons by electrical stimulation of the pituitary stalk
did not elicit signifi cant dendritic release of OT in virgin rats. Thus,
although it is not known if a backpropagated AP can effectively