Leitz dmrd microscope

Animals were premedicated with ketamine hydrochloride (Vetalar; 0.05 ml of a 100 mg solution, i.m.; GE Healthcare and Upjohn) before being euthanized with pentobarbitol sodium (Dolethal; 1 ml of a 200 mg/ml solution, i.v.; Merial Animal Health). Animals were then perfused transcardially with 500 ml of 0.1 m PBS, followed by 500 ml of 4% paraformaldehyde fixative solution over ∼15 min. The brain was removed and left in the 4% paraformaldehyde fixative solution overnight before being transferred to 30% sucrose solution for at least 48 h. Brains were then sectioned on a freezing microtome (coronal sections; 60 μm), mounted on slides, and stained with cresyl fast violet. The sections were viewed under a Leitz DMRD microscope (Leica Microsystems). The cannula locations for each animal were schematized onto drawings of standard marmoset brain coronal sections and composite diagrams were then made to illustrate the extent of overlap between animals.

Animals were pre-medicated with ketamine hydrochloride before being euthanized with pentobarbital sodium (Dolethal; 1ml of a 200mg/mL solution, i.e.; Merial Animal Health, Essex, UK). Animals were then perfused transcardially with 300ml 0.1M PBS, followed by 300ml of 4% paraformaldehyde fixative solution over approximately 15 min. The brain was removed and left in the 4% paraformaldehyde fixative solution overnight before being transferred to 0.01M PBS-azide solution for at least 48 hours. Finally, the brains were transferred to 30% sucrose solution for at least 48 hours for cryoprotection. Brains were then sectioned on a freezing microtome (coronal sections; 40-60μm), mounted on slides and stained with cresyl-violet. The sections were viewed under a Leitz DMRD microscope (Leica Microsystems, Wetzlar, Germany). The cannula locations for each animal were schematized onto drawings of standard marmoset brain coronal sections together with estimated infusion spread (Figure S1A) which was estimated at 0.5-1.0mm (based on Allen et al., 2008 (link)).

At the end of the experiment, all monkeys were sedated with ketamine hydrochloride (Pharmacia and Upjohn, 0.05 mL of a 100 mg/mL solution, i.m.) and humanely euthanized with Euthatal (1 mL of a 200 mg/mL solution, pentobarbital sodium; Merial Animal Health Ltd; i.v.) before being perfused transcardially with 400 mL of 0.1 M phosphate-buffered saline (PBS), followed by 400 mL of 4% paraformaldehyde fixative solution over approximately 15 minutes. The cannulae and dental cement were carefully removed. After the brain was removed, it was left in the 4% paraformaldehyde fixative solution overnight, before being transferred to 0.01M PBS-azide solution for at least 48 hours and then transferred to 30% sucrose solution for a further 48 hours for cryoprotection. Brains were sectioned on a freezing microtome (coronal sections; 40-60mm), mounted on slides and stained with cresyl violet. The sections were viewed under a Leitz DMRD microscope (Leica Microsystems, Wetzlar, Germany). The cannula locations for each animal were represented on schematized coronal sections of the marmoset brain (Figure 2). Before euthanasia, some animals underwent infusions of drugs for c-fos verification and one animal underwent an anatomical tract-tracing study.