Biology Reference
In-Depth Information
this behavioral experiment was treated as described above and
injected with 1
-ChR2(H134R)-mCherry in
M1 cortex [AP 1 mm; ML 1 mm (left); DV 1.5 mm]. The proce-
dure detailed below was performed 4 weeks after injection.
μ
l AAV2/5-CaMKII
α
1. Position the rotary joint above the center of the open fi eld
chamber and next to the video tracking camera.
2. Connect the long (5 m) patch cord to the laser fi ber coupler
on one end and to the rotary joint at the other end.
3. Connect the second fi beroptic patch-cord from the rotary
joint so that the bare ferrule side terminates at the level of the
open fi eld chamber.
4. Remove the mouse from the home cage and attach the fi ber-
optic patch-cord ferrule to the IFL.
5. Place the mouse in the center of the open fi eld and initiate the
behavioral recording session.
6. At defi ned times, light can be delivered through the fi beroptic
cables by triggering the laser through TTL pulses from the
pattern generator. Depending on the laser type, digital or ana-
log modulation can be applied to control the light power out-
put ( see Note 10 ).
7. At the end of the experimental session, remove the mouse
from the arena, disconnect the fi beroptic patch-cord from the
IFL, and return the mouse to the home cage.
2.6 Procedure
In the protocol section, we describe a procedure for viral transduc-
tion of primary motor cortex pyramidal neurons with
ChR2(H134R)-mCherry, followed by an optical fi ber implanta-
tion for in vivo modulation of neuronal activity. To test the effect
of optogenetic activation of primary motor cortex pyramidal neu-
rons in a freely moving mouse, we placed the implanted mouse in
an open fi eld chamber and recorded the position of the mouse
during a behavioral session that lasted 30 min. At defi ned times, we
applied 447 nm light at different frequencies and analyzed the
changes in the direction of locomotion compared with baseline
(light-off) periods. The raw tracking data were imported into
Matlab and analyzed offl ine for the direction of locomotion. The
position of the mouse was sampled in 0.5 s intervals. We measured
the angle of the third point relative to the line defi ned by the fi rst
and second points. The resulting vector showed an average angle
of 28° towards the contralateral side during episodes of optical
stimulation (with respect to the site of optical stimulation). While
the angle of this trajectory is clearly dependent on the sampling
frequency, the resulting estimated average radius of 6.3 cm is stable
over a wide range of sampling intervals. We demonstrate that
2.7 Summary
and Expected Results
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