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.