Viral Vectors for Optogenetics of Hypothalamic Neuropeptides - Viral Vector Approaches in Neurobiology and Brain Diseases - page 309

ber of intact connections. This can be of particular importance
when studying indirect effects such as the ones we studied in
the central medial amygdala (see [ 8 ]). In addition, it allows a
high enough number of ChR2-axons for experiments that rely
on to direct stimulation of axonal release.
4. If the experimental design allows it, keep some slice(s) from
the region where the virus was originally injected (in our case
PVN, AN, and SON) for further examination in order to ver-
ify suffi cient expression of the construct at the level of the cell
body. Suffi cient expression of the fl uorescent reporter gene
will be a fi rst criterion of selection of cells for patch-clamp
recordings (to be observed under a fl uorescence microscope).
Subsequent whole-cell patch-clamp recordings of neurons will
show their sensitivity to blue light refl ecting the levels of ChR2
expression (see below).
5. Place the slices in a dark interface chamber with circulating
ACSF (1.5 ml/min) saturated with oxycarbon. This particular
disposition allows a survival of the slices for 12 h and more.

1. When using channelrhodopsin, it is critically important to
assess the level of expression of the protein. The fi rst step is
to visualize the expressing cell through fl uorescent imaging.
A second step, more important, is to ensure that the expres-
sion is high enough to induce activation of the cell. To this
purpose, blue light pulses must induce repeatable cell depolar-
izations that are able to reliably induce action potentials (APs).
For OT cells in the hypothalamus, we obtained 90 % success
rate in inducing APs with pulses of 10 ms at 10 Hz as mea-
sured in whole-cell current-clamp measurements. This success
rate decreased to 50 % when pulses were applied at 30 Hz fre-
quencies—though the total number of evoked APs in the same
time period was increased. Under voltage clamp, this trans-
lated into a transient inward current of ~300 pA and a sus-
tained inward current of ~200 pA for each pulse of blue light
(473 nm and 5 mW/mm 2 ).
2. When suffi cient levels of ChR2 have been assessed in the cell
body, axonal expression of ChR2 should also be verifi ed in the
region in which release is to be induced. The presence of fl uo-
rescent fi bers should, in principle, indicate the simultaneous
expression of channelrhodopsin in these structures and hence
their sensitivity to stimulation with blue light.
3. When inducing the release of the endogenous hypothalamic
neuropeptide through activation of either the soma or the
neurite, the potential corelease of the neuropeptide with
another peptide or neurotransmitter should always be consid-
ered. Another important source of confusion is the site of
action of the secreted neuropeptide. For example, it is well

2.3.4 In vitro Recordings

General Considerations