Stereotaxic frame

The surgery was performed on rats anesthetized with isoflurane under aseptic conditions with body temperature maintained at 37 °C using a rectal probe and feedback-controlled heating pad (Harvard Apparatus). Rats were positioned in a stereotaxic frame (Harvard Apparatus) and the skull exposed with a midline incision. A cranial burr hole was drilled above the right lateral ventricle (0.6 mm posterior and 1.6 mm lateral to bregma), after which the rats were removed from the stereotaxic frame, the femoral artery catheterized, and approximately 300 µl blood was collected (the control rats underwent sham operation). Immediately thereafter, 200 µl of this autologous blood sample (or saline) was manually injected over the course of 15 min via a 27-gauge needle inserted stereotaxically into the burr hole in the right lateral ventricle (4.5 mm ventral) [29 (link)] and expected to spread throughout the ventricular system containing approximately 180 µl CSF in rats of the chosen species and age (181 ± 18 µl, n = 3, quantified by MRI, see below). The needle was kept in place for 5 min before retraction to prevent backflow. The skin incisions were closed with sutures and the rats were allowed to recover before returning to the housing facility.

Burprenorphine was delivered (I.P 0.05 mg/kg) 30 min before surgery. The animals were deeply anesthetized with isoflurane (1.5–2.5%), and the body temperature maintained at 37°C using a heating pad. The animals were fixed in a stereotaxic frame (Harvard Apparatus, Holliston, MA) and a small craniotomy (0.5 mm) was made unilaterally over the mPFC (AP 3.0–3.3 mm, ML 0.5–0.8 mm). The virus (AAV5-Ef1a-DIO-hChR2(H134R)-mCherry, UNC Gene Therapy Center, #AV4314B, 4x10¹² virus molecules/ml) was delivered by a glass capillary attached to a motorized Quintessential Stereotaxic Injector (Stoelting, Wood Dale, IL, USA) at rate of 0.05–0.1 μL min⁻¹. Injection in the mPFC: half the volume (0.5 μL) was injected at DV -2.0 mm. The capillary was thereafter held in place for 5 min before being slowly lowered to DV -3.0 mm, at which the rest of the virus (0.5 μL) was injected. The pipette was thereafter held in place for 10 min before being slowly retracted from the brain. The incision was closed with stitches (Ethicon, USA), and postoperative caprofren (5 mg/kg) was administered every 24 h for 48 h.

Burprenorphine was delivered (I.P 0.05 mg/kg) 30 min before surgery. The animals were deeply anesthetized with isoflurane (1.5–2.5%), and the body temperature maintained at 37°C using a heating pad. The animals were fixed in a stereotaxic frame (Harvard Apparatus, Holliston, MA) and a small craniotomy (1.0–1.5 mm diameter) was made unilaterally over the mPFC (AP 3.0–3.3 mm, ML 0.5–0.8 mm). The microdrive was positioned above the craniotomy with the optical fiber and tetrodes gradually lowered to the PrL (2.5 mm ventral to brain surface), aimed at layer 5/6. Five miniature anchoring screws were used to attach the microdrive to the skull (one contralateral to the microdrive, and two rows of two screws on the anterior and posterior part of the parietal bone). One Teflon coated stainless steel wire (0.005 inch bare, A-M systems) from the electrode interface board (EIB) of the microdrive was connected to the screws for grounding. The microdrive was secured onto the skull using dental adhesive cement (Super Bond C&B, Sun Medical). 3 rats were implanted in the right hemisphere and 4 rats in the left hemisphere. After surgery the animals were single housed. Postoperative carpofen (5 mg/kg) was administered every 24 h for 48 h.

Euploid and Ts65Dn young adult mice (2 to 2.5 month-old) were anesthetized with a combination of 1 mg/Kg medetomidine (Domtor, Pfizer) and 75 mg/Kg ketamine (Imalgene 500, Merial), and immobilized in a stereotaxic frame (Harvard Apparatus). Bilateral injections were performed at the level of the ventral hippocampus at the following coordinates relative to bregma (anterior-posterior = −3.3 mm, medial-lateral = +/− 3 mm, dorso-ventral = −3.3 mm and −2.3 mm) using a 5 μl Hamilton syringe. Up to 1008 transducing units (3 μl of viral suspensions of Lv-Control or Lv-miR155-802T) were injected into each hemisphere at a rate of 0.2 μl/min, under the precise control of an infusion pump (Ultramicropump, World Precision Instruments). The needle was left in place for 5 min after injection and then slowly retracted from the brain. Before complete withdrawal, the needle was allowed to dwell for an additional 5 min. After surgical intervention, the animals were injected subcutaneously with a dose of 2 mg/kg of atipamezole (Antisedan, Pfizer) for anaesthetic reversal. The analgesic, buprenorphine (Buprex, ScheringPlough) was also administered intraperitoneally at a dose of 0.05 mg/Kg twice a day for the following 72 h after intervention. Mice were euthanized and the hippocampus dissected at day 23 after infusion.

Mice were anesthetized with isoflurane (2%) and placed into a stereotaxic frame (Harvard Apparatus). Before the first incision, the analgesic Buprenorphine (0.1 mg kg^–1) and local analgesic Xylocain/Lidocain (4 mg kg^–1) was administered subcutaneously. The body temperature of the mice was maintained at 36 °C with a feedback-controlled heating pad. For viral injections a micropipette attached on a Quintessential Stereotaxic Injector (Stoelting) was used. Injections were done with a speed of 50 nl min^–1. The injection pipette was held in place for 5 min after the injection before being slowly (100 µm s^–1) retracted from the brain. The analgesics Carprofen (5 mg kg^–1) was given at the end of the surgery, followed by a second dose 18–24 h after surgery.

SOM- cre mice were anesthetized with isoflurane and placed in a stereotaxic frame (Harvard Apparatus, Holliston, MA). A Quintessential Stereotaxic Injector (Stoelting, Wood Dale, IL) was used to inject 0.5 µl of AAV5-EF1a-DIO-EYFP (#27056, Addgene) into the striatum (+0.5 AP, 2 ML, 2.5 DV) at a speed of 0.1 µl/min. The pipette was held in place for at least 5 min after the injection. Following the surgery, the mice were given analgesics (buprenorphine, 0.08 mg/kg, i.p.).

Mice underwent surgery for injection of AAV9-CB7∼Cl-mCherry or -GFP virus into the PBN (Fig. 1). Retrograde transport, genomic incorporation, and subsequent expression of the fluorescent protein in projection neurons allowed targeted patch clamp recordings.^{46 (link)} Briefly, mice were anaesthetised with isoflurane (5% induction, 1.5%-2% maintenance) and secured in a stereotaxic frame (Harvard Apparatus, MA). Craniotomies provided access for unilateral (anatomical experiments) or bilateral (electrophysiology) PBN injection of ∼700 nL of virus through a picospritzer (PV820, WPI, FL). Injections were made over 5 minutes at stereotaxic coordinates of 5.25 mm posterior to bregma, 1.2 mm lateral to the midline, and at a depth of 3.8 mm from the skull surface according to The Mouse Brain Atlas (Paxinos and Franklin 2001). The pipette was left in place for 7 to 10 minutes after the injection to minimise drawing the virus sample along the pipette track. We adopted a 2 to 4 week postinjection recovery time to allow optimal retrograde labelling of projection neurons before spinal cord slices were prepared.^{28 (link)} All animals made an uneventful recovery and showed no overt disturbances to behaviour.