Anterograde Tracing of Supramamillary Nuclei

The anterograde tracer, Biotin-Dextran-Amine (BDA 2 %; MW 3,000; Invitrogen, Carlsbad, CA), was injected under stereotaxic condition into the SuML and SuMM according to previously described protocol (Soussi et al. 2010 (link)). Control (n = 6) and pilocarpine-treated rats at 2 and 4 months (n = 6) were anesthetized with chloral hydrate (400 mg/kg; i.p.; Fisher Scientific, Loughborough, UK), secured in a stereotaxic frame (David Kopf, Tujunga, CA) and pressure-injected with 300 nL (30 nL/min during 10 min) of BDA either into the SuML at the following coordinates: 3.9–4 mm posterior to bregma (AP), 0.5–0.7 mm lateral to the midline (L) and 8.9–9 mm ventral to the cortical surface (V) or into the SuMM at the following coordinates: AP = 4.3–4.7 mm L = 0.2 mm and V = 8.8 mm. After the completion of injection procedures, the syringe was removed and the skin sutured. The animals were treated with topical anesthetic and then returned to their home cages for a survival period of 8–10 days to allow proper anterograde transport of the tracer.