Surgery
Prior to surgery, all rats were anaesthetised with an isoflurane-oxygen
mixture (5% induction, 1.5-2.5% maintenance). Then, each rat was
placed in a stereotaxic frame (David Kopf Instruments, CA, USA), so that
the skull was flat, with respect to the horizontal plane.
Chloramphenicol 0.5% eye-gel was applied, meloxicam (0.06ml) was
administered subcutaneously for analgesic purposes, and lidocaine (0.1ml
of 20mg/ml solution) was applied topically to the incision site. Next, a
bilateral craniotomy was performed above the dorsal subiculum, and
either pAAV-CaMKIIa- hM4D(Gi)-mCherry (AAV5) (iDREADD)(Titer:
2.6x10^13GC/ml, lot:v102676; Addgene, MA, USA) or pAAV-CaMKIIa-GFP
(AAV5)(Titer: 4.3x 10^12GC/ml, lot: v5894, Addgene, MA,
USA)(GFP-control) virus was injected bilaterally into the dorsal
subiculum.
In both cohorts, 0.6µl of the
viral construct was injected in the anterior subiculum injection site
and 0.4µl into the more posterior site. The injection coordinates, with
respect to bregma were as follows: Anterior . Cohort 1: AP:
-5.9mm, ML: ±2.9mm, DV: -2.6mm; Cohort 2: AP: -5.9mm, ML: ±2.7mm, DV:
-2.4mm; Posterior . Cohort 1: AP: -6.2mm, ML: ± 3.2mm, DV: -2.5mm;
Cohort 2: AP: -6.2mm, ML: ± 3.0mm, DV: -2.3mm), respectively. The very
slight changes in coordinates reflected individual preferences of two
researchers, based on pilot experiments. All injections were made
vertically using a 10µl Hamilton Syringe attached to a movable arm. A
micro-syringe pump (World Precision Instruments, Florida, USA)
controlled the injection, with the flow rate set at 150ηl/min. The
injection needle was left in situ for further 5 minutes, before
retracting it. The order of the iDREADDs and GFP injections was
randomized, so that animals were randomly allocated to either group.
During the same surgeries, pairs of cannulas were implanted into the
left and right retrosplenial cortex. One cannula pair (1.5mm length x
1.2mm separation, 26-gauge, PlasticsOne, Virginia, USA) was implanted
into the anterior portion of the retrosplenial cortex (from bregma; AP:
-2.5 mm, ML: ± 0.6 mm, DV: -1.5mm), the other cannula pair (1.7mm length
x 1.4mm separation; 26-gauge, Plastic One, Virginia, USA) was implanted
into the posterior retrosplenial cortex (AP: -6.0mm, ML: ± 0.7mm, DV:
-1.7mm). The implantation coordinates for both cohorts remained the
same. The cannulas were held in place with bone cement (Zimmer Biomet,
Swindon, UK) and anchored to the skull with four screws (Precision
Technology Supplies, Uckfield, UK). Dummy cannulas were inserted into
the guide cannulas to prevent blocking and were secured in place with
aluminium dust caps. The analgesic Marcaine Polyamps (AstraZeneca, UK)
and antibiotic powder (Clindamycin, Pfizer, UK) were applied to the
surgical site. All animals were subcutaneously administered 5ml
glucose-saline solution for fluid replacement, prior to placing them in
a recovery chamber. Once the animals regained consciousness, they were
returned to their home cage and closely monitored.