Biomedical Engineering Reference
In-Depth Information
power laser can cause astrocytic Ca
2
+
oscillation in vivo
(9)
.The
average laser power that was used for detecting astrocytic Ca
2
+
signaling in barrel cortex layer 2 was less than 30 mV under the
objective.
4.4. Whisker
Stimulation
Whiskers were stimulated by air puffs
(48)
. Briefly, whiskers on
one side of the snout were trimmed to a 10-mm length. Air
puffs consisted of pulses of compressed air, which were generated
from a high pressure nitrogen tank and controlled by picospritzer
(General Valve) at a pressure of 10 psi and delivered by a thick wall
plastic tubing (4.5 mm outer-diameter, 2.5 mm inner-diameter).
The tube was placed parallel to the left side of the mouse snout
and 10-20 mm away from the C6 whisker. The direction of air
flow was from rostral to caudal along the whisker row. This proto-
col will stimulate most of the vibrissae on one side of the whisker
pad (at least three rows of whiskers were consistently activated by
air puffs) (
Fig. 5.2)
. The frequency of air puffs was set to 1, 3,
5, 7 or 10 Hz and controlled by Master-8 (A.M.P.I). Pulse width
in all experiments was 10 ms. The duration of stimulation was
adjusted from 1 to 60 s in different experiments. Single whisker
stimulation was not attempted at this point, but in future studies,
it will be intriguing to deflect a principle whisker using mechanic
stimulation
(49)
and image the astrocyte Ca
2
+
responses in its
related barrel column and the nearby columns.
4.5.
Electrophysiological
Recording
LFP recordings were obtained from layer 2 of barrel cor-
tex (100-150
m below the pial surface) by a patch pipette
(TW100F-4, WPI; outer diameter, 1.0 mm; inner diameter, 0.75
mm; tip diameter,
μ
∼
μ
), contain-
ing 0.2% Texas red-dextran (77,000 molecular weight, Sigma)
in aCSF. LFP signals were amplified using Axopatch 700B and
pCLAMP 8.2 program with DigiData 1332A interface (Axon
Instruments). The signals were bandpass filtered at (1-100 Hz)
and digitized at 10 kHz.
3
m; tip resistance 3-5 M
5. Properties of
Astrocytic Ca
2
+
Signaling Evoked
by Whisker
Stimulation
Astrocytes in barrel cortex layer 2/3 of adult mouse were labeled
with Ca
2
+
indicator fluo4-AM. Recording electrode was inserted
into the field of imaging to detect LFP activity (
Fig. 5.3A
).
Although increased neuronal activity in layer 2/3 of barrel cortex
can be detected as early as 10-12 ms after whisker deflection
(50)
,
increases in astrocyte somatic Ca
2
+
3safter
the onset of whisker stimulation
(9)
. The onset of the increase in
astrocytic Ca
2
+
was defined as the earliest time point at which the
were delayed by
∼
