Biotin dextran amine

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.

Four adult mice (approximately three to four months old) were anesthetized and then, using a stereotaxic apparatus (Kopf Instruments), were intracerebrally injected with the anterograde tracer biotin dextran amine (BDA; 10 kDa; 10% in 0.1 m PB; Invitrogen) in the supramammillary nucleus (bregma −2.80 mm, lateral 0 mm, depth 4.3 mm). Coordinates were calculated according to the Paxinos’ Atlas (Paxinos and Franklin, 2012 ). The tracer was delivered by iontophoresis via 7-s on/off 5- to 10-μA current pulses for 15 min using a borosilicate micropipette. After 5–7 d of survival, animals were transcardially perfused with the glutaraldehyde fixative as described previously. A parallel series of sections taken from each animal (one in six sections) was incubated in avidin-biotin complex (ABC; 1:200, DAKO) in PB containing 0.1% Triton X-100 and developed using nickel intensified diaminobenzidine (DAB) to visualize the injection site and the anterograde labeling.