Real-time ultrasound imaging (Vevo 1100 System,
Fujifilm Visualsonics) allowed for targeted delivery
of the viral mixture to specific areas. For imaging,
the ultrasound probe (MS-550S) was lowered to
be in close contact with the gel to allow visualization of the targeted structures, and was kept
in place for the whole duration of the procedure
via the VEVO injection mount (VEVO Imaging
Station. Imaging in B-Mode, frequency: 40 MHz;
power: 100%; gain: 29 Db; dynamic range: 60 Db).
Target regions were identified by structural landmarks: the hippocampus was identified by the
cytoarchitecture of CA3 and the appearance of
the lateral ventricle (target area for injection was
comparable to a coronal section at -1.46 mm from
bregma in the adult animal (48)); the MEC and
LEC were identified by the appearance of the
aqueduct of Sylvius and the lateral sinus (target
area for injection was comparable to a coronal
section at -4.72 mm from bregma in the adult
A viral mixture (250 ± 50 nl per injection) was
injected in the target regions via beveled glass
micropipettes (Origio, custom made. Outer tip
opening: 200 mm. Inner tip opening: 50 mm) with
a pressure-pulse system (Visualsonics, 5 pulses,
50 nl per pulse). The anatomical specificity of
the infection was verified by imaging serial sections of the infected hemispheres after experiment completion.
Virus injection in the embryo
Pregnant females were subjected to the viral in-
jection on a specific day of gestation, from E10 to
E17. To achieve precise monitoring of gestation,
a mating trio (one male and two females) was
allowed to interact for a limited period of time
(24 hours), after which the male was removed
from the cage to prevent further mating. E1 was
defined as the day on which the male was re-
moved (vaginal plug could be observed in most
of the cases). A longitudinal incision was per-
formed on the shaved skin and peritoneum of
the isoflurane anaesthetized female to reach the
uterine horns. The abdominal cavity was kept
irrigated with warm saline solution (0.9% NaCl,
39°C) for the entire procedure. Single embryos
were transferred one-by-one to a custom made,
sterile surgical chamber to allow for viral injec-
tion. Pre-heated ultrasound gel (39°C, Aquasonic
100, Parker) was generously applied to the embryos.
Real-time ultrasound imaging (parameters as
above) allowed for targeted delivery of the viral
mixture to the lateral ventricle of the developing
brain. The viral solution (containing AAV1-CaMKII-
Cre; 150–350 nl per injection) was applied as pre-
viously described (UPenn Vector Core, University
of Pennsylvania, Perelman School of Medicine).
After injection, the ultrasound gel was removed
with sterile gauze and the embryo placed back
into the abdominal cavity. This procedure was
repeated for all the other embryos in the litter.
After injection of the last embryo, two indepen-
dent sets of stitches were applied to the perito-
neum and abdominal skin. The total length of
the procedure was limited to less than 90 min
to avoid unnecessary stress to the mother and
the embryos. 80% of the injected embryos sur-
vived injection and delivery, and could be utilized
for further studies.
On P11 or P14, a longitudinal incision was performed on the shaved skin of the isoflurane
anaesthetized pup’s back in order to allow for the
insertion of a minipump subcutaneously. Warm
saline solution (0.9% NaCl, 39°C) was injected
subcutaneously in the area of the incision at
multiple times during the minipump implant. The
osmotic minipump (Alzet, 1007D: flow of 0.5 ml
per hour for up to 7 days) filled with 100 ml of CNO
solution (1 mg/ml in saline solution, Sigma) was
then placed through the incision to rest comfortably on the mouse’s back. No impairment of normal behavior as a result of discomfort from the
implant could be observed in the days following
the procedure. CNO delivery was protracted over
the course of several days during maturation, with
a steady flow of 0.5 ml per hour according to the
manufacturer’s specification (Alzet). In a subset
of P14-implanted animals (14 subjects), the minipump was surgically removed at P20 through a
second incision. Seven subjects were perfused
after 3 days of recovery (P23), while another 7 were
perfused after 6 days of recovery (P26) and processed for further analysis.
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