Viral Vectors for Optogenetics of Hypothalamic Neuropeptides - Viral Vector Approaches in Neurobiology and Brain Diseases - page 310

known that vasopressin can bind to both oxytocin and vaso-
pressin receptors. Therefore, one should always assess which
receptor is indeed activated following activation by blue light.
These two points can easily be addressed by pharmacological
experiments using appropriate highly selective antagonists.
4. Receptors for hypothalamic peptides are often G-protein
coupled. Thus, it is crucial to avoid washout of the intracellular
signal transduction machinery when studying the direct effects
of endogenous release. This can be done by recording in cell-
attached confi guration, or by using a specifi c intrapipette
solution when recording in whole-cell confi guration.

1. Once the slice is placed in the recording chamber, focus the
blue light on the region of interest. When using a mercury
lamp, the diameter can be restricted by reducing the opening
of the diaphragm in order to stimulate the minimum amount
of fi bers/cells.
2. Establish a base line of the responses by doing repeated stimu-
lations. Indeed, desensitization of the receptor can be a serious
issue when studying the pharmacology of oxytocin [ 65 ].
3. Fill the pipette with biocytin if further histological investigation
of the neurons is foreseen.

Step by Step

2.4 In Vivo
Optogenetics
(See Fig. 2 )

Since blue light is strongly scattered by brain tissue [ 21 ],
illumination from short distances can already dramatically attenu-
ate its power. Indeed, Yizhar et al. [ 1 ] showed that power emitted
through the fi ber (see below) is already after half a millimeter
reduced to less than one percent of the initial value at the fi ber tip.
Although this can be an advantage for restricting the area of the
stimulation in comparison with drug injection, it should also be
carefully considered during the surgery in order to correctly target
the very small region of interest.

2.4.1 Material

General Considerations

1. Intracerebral cannulae with appropriate diameter for optical
fi bers (C313G-SPC, outer diameter: 0.71 mm, inner diame-
ter: 0.390 mm, PlasticOne, Roanoke, Vancouver). The length
of the cannulae should be chosen such that the tip of the can-
nula does not approach the region of interest closer than
1 mm in order to avoid possible tissue damage.
2. Light emitter. Light stimulation can be provided by a blue
laser that is capable of delivering short pulses (2-10 ms) with
adequate frequency (at least up to 30 Hz for hypothalamic
neurons) (

Detailed Material

473 nm, output of 50 mW DreamLasers,
Shanghai, China).
3. Optical fi ber. Light from the source of origin (the laser in our
case) is transmitted through optical fi bers (BFL37-200;
ThorLabs, Newton, New Jersey; 200 um diameter) that are

λ