Model 5000

The rats were ∼ 3 months old at the time of surgery. All rats were anaesthetised with isoflurane gas. They were then placed in a stereotaxic frame with the incisor bar set at − 3.3 mm, and given analgesia in the form of 0.1 mg/kg Metacam (Boehringer Ingelheim Vetmedica, Germany) administered subcutaneously. To expose the skull, a midline sagittal incision was made in the scalp, and the skin was reflected. A craniotomy was made above the injection sites, and the dura was cut to expose the cortex. The hippocampal lesions (n = 22) were made with injections of ibotenic acid (Biosearch Technologies, San Rafael, CA, USA) diluted to 63 mm in phosphate-buffered saline (PBS; 0.1 m, pH 7.4). The ibotenic acid was administered via a 2-μm Hamilton syringe connected to a microinjector (Model 5000; Kopf Instruments) set at a rate of 0.1 μL/min, with a subsequent diffusion time of 2 min. The rats received 14 injections into each hemisphere [for coordinates and volumes, see Iordanova et al. (2009 (link))]. The surgical control rats (n = 20) were treated in the same way until the dura was exposed. While nothing was infused into the brain, the dura was pierced 14 times per hemisphere with a 25-gauge Microlance needle (Becton Dickinson, Drogheda, Ireland).

Animals were fully anaesthetized with pentobarbital anesthesia (50 mg/kg, i.p., Vetbutal, Biowet Pulawy, Poland) and atropine sulfate (0.25 mg/kg, s.c, Polfa, Poland) and placed in a stereotactic apparatus (Kopf Instruments, USA). The skull was exposed by a midline incision of the skin and a hole was drilled above the lesion site. The neurotoxin 6-OHDA (6-hydroxydopamine HCl, Sigma–Aldrich, Poland) was injected into the right SNpc in a volume of 4 μl (3 μg/μl dissolved in 0.9% NaCl containing 0.1% ascorbic acid). The following coordinates from the rat stereotactic atlas (Paxinos and Watson 2007 ) were used (in reference to bregma): anteroposterior (AP) – 5.3, lateral (L) – 2.4, dorsoventral (DV) – 7.5. Injections were performed using a microsyringe with a 26-gauge needle (Hamilton, USA) that was attached to a microinjection unit (Model 5000, Kopf Instruments, USA). The injection rate was 0.5 μl/min and the cannula was left in place for additional 5 min after injection to allow for diffusion into the tissue. To protect noradrenergic neurons from damage, animals received an intraperitoneal injection with the noradrenaline reuptake inhibitor desipramine (25 mg/kg, Sigma-Aldrich, Poland) 30 min prior to neurotoxin injection (Fulceri et al. 2006 (link)). Sham-lesioned controls underwent the same procedure but received vehicle instead of 6-OHDA.