Multimodal Gene Delivery and Neuromodulation in Rats

The first group of animals consisted of 7 rats of which 4 rats were bilaterally injected with 1 µl of 1.0×10^∧9 genomic copies/µl of CAV-2 (IGMM, France) and 3 rats with 1 µl of 5.0×10^∧8 genomic copies/µl of CAV-2 in the Acb (from bregma: anterioposterior: +1.2 mm, medio-lateral: ±2.8 mm; dorso-ventral: −7,5 mm, at an angle of 10°), using a stereotactic apparatus. Next, all rats were bilaterally injected with 1 µl of 1.0×10^∧9 genomic copies/µl of AAV-hSyn-DIO-hM₃D(G_q)-mCherry (UNC Vector Core, USA) in the VTA (from bregma: anterio-posterior: −5.4 mm, medio-lateral: ±2.2 mm, dorso-ventral: −8.9 mm, at an angle of 10°). As there was no difference observable in VTA hM₃D(G_q)-mCherry expression or behavioral measures between the CAV-2 titers, the groups were considered to be equal and combined for statistical analyses. Infusions were performed under fentanyl/fluanisone (0.315 mg/kg fentanyl, 10 mg/kg fluanisone, i.m., Hypnorm, Janssen Pharmaceutica, Belgium) and midazolam (2.5 mg/kg, i.p., Actavis, the Netherlands) anesthesia. Xylocaine was sprayed on the skull to provide local anesthesia (Lidocaine 100 mg/ml, AstraZeneca BV, the Netherlands). A transmitter for the recording of locomotor activity (TA10TA-F40, Data Science International, USA) was placed in the abdominal cavity as well. All rats received three daily peri-surgical injections of carprofen (5 mg/kg, s.c., Carporal, AST Farma BV, the Netherlands) starting at the day of surgery.
A second group of 8 rats underwent identical surgery procedures but received additional guide cannulae (24 gauge; Cooper’s Needleworks, UK) in the Acb (from bregma: anterioposterior: +1.2 mm, medio-lateral: ±2.8 mm; dorso-ventral: −6,5 mm, at an angle of 10°) as well. Cannulae were secured on the skull with stainless steel screw and dental acrylic and stainless steel stylets (29 gauge) were inserted in the guide cannulae to ensure patency.