 the following questions. Is the mTOR pathway activated when mossy fiber sprouting occurs after status epilepticus? Does prolonged, continuous, focal infusion of rapamycin suppress development of mossy fiber sprouting? If so, does mossy fiber sprouting remain suppressed after rapamycin treatment ceases? And does rapamycin infusion reverse previously established mossy fiber sprouting?
All experiments were approved by the Stanford University Institutional Animal Care and Use Committee and performed in accordance with National Institutes of Health Guide for the Care and Use of Laboratory Animals. Pilocarpine treatment was performed as described previously (Buckmaster, 2004a). Briefly, male Sprague Dawley rats (34 ± 1 d old; range 27–45) were treated with atropine methylbromide (5 mg/kg, i.p.), then 20 min later with pilocarpine hydrochloride (380 mg/kg, i.p.) to induce status epilepticus. All chemicals were from Sigma-Aldrich unless specified otherwise. After 2 h of status epilepticus, convulsions were suppressed with diazepam (10 mg/kg, i.p., repeated as needed; Hospira), and lactated Ringer's solution was administered subcutaneously. All rats in this study experienced at least 2 h of status epilepticus.
Osmotic pumps and cannulae were implanted to focally and continuously deliver rapamycin (Alexis Biochemicals or LC Laboratories) to the dorsal, left dentate gyrus. Rats were anesthetized with 2% isoflurane (Baxter). Body temperature was monitored and controlled with a heating pad with feedback control. Rats were placed in a stereotaxic apparatus, and their head and dorsal neck were prepared for aseptic surgery. A scalp incision was made, and a <1-mm-diameter hole was drilled through the skull, 4.6 mm caudal and 2.8 mm left of bregma. A 3.5-mm-long 28 gauge cannula (Alzet brain infusion kit II; Durect Corporation) was inserted and secured to the skull with cranioplastic cement and a jeweler's screw. An osmotic pump (model 2004; Durect Corporation) and tubing leading to the cannula were implanted subcutaneously over the back. The manufacturer's specifications indicate that at body temperature (37°C), model 2004 pumps deliver 0.25 ± 0.05 μl/h for at least 28 d. Pumps contained vehicle solution consisting of 50% DMSO, 15% ethanol, and 0.1% fluorescein (Invitrogen) with or without rapamycin (0.01–10 mm). In most experiments, infusions began 1–8 h after administering the first dose of diazepam, which was 2 h after onset of status epilepticus. In one experiment, surgery and rapamycin infusion was delayed until 2 months after status epilepticus. Rapamycin was infused for 1 or 2 months. For 2-month-long infusions, pumps were replaced after 1 month. In most experiments, rats were perfused immediately after 1- or 2-month-long infusions. In one experiment, perfusion was delayed until 2 months after rapamycin infusion ceased.
Rats were perfused and hippocampi sectioned and processed as described previously (Buckmaster, 2004b). Briefly, rats were killed by urethane overdose (2 g/kg, i.p.), perfused through the ascending aorta at 30 ml/min for 2 min with 0.9% sodium chloride, 5 min with 0.37% sodium sulfide, 1 min with 0.9% sodium chloride, and 30 min with 4% paraformaldehyde in 0.1 m phosphate buffer (PB, pH 7.4). Brains postfixed overnight at 4°C. Then, both hippocampi were isolated, cryoprotected in 30% sucrose in 0.1 m PB, gently straightened, frozen, and sectioned transversely with a microtome set at 30 μm. Starting at a random point near the septal pole, a 1-in-6 series of sections from each hippocampus was mounted, dried, and developed for 45 min in 120 ml of 50% gum arabic, 20 ml of 2 m citrate buffer, 60 ml of 0.5 m hydroquinone, and 1 ml of 19% silver nitrate. Infused and contralateral noninfused hippocampi from the same animal developed together. An adjacent 1-in-6 series of sections was processed for Nissl staining with 0.25% thionin. A third 1-in-6 series was mounted, dried, coverslipped with Vectashield (Vector Laboratories), and checked for fluorescein labeling to verify delivery of pump contents in infused hippocampi.
Mossy fiber sprouting was measured as described previously (Buckmaster and Dudek, 1997; Buckmaster, 2004b). Briefly, sections were analyzed using a light microscope equipped with a 10× objective, Lucivid, and Neurolucida software (MBF Bioscience). An investigator blind to the rats' treatment made contours around the granule cell layer + molecular layer and the Timm-positive parts of the granule cell layer + molecular layer. Figure 1 illustrates a typical example of contours drawn on a Timm-stained section of dentate gyrus. Density of Timm staining within Timm-positive contours was measured using identical camera and microscope settings for all samples. Brightness was measured and expressed on an optical density scale from 0 to 1, in which 0 was the minimal density value when no tissue was in the light path, and 1 was the maximal density value when the microscope light source was turned off. Extent of mossy fiber sprouting varies among individual rats treated systemically with chemoconvulsants (Buckmaster and Dudek, 1997). To avoid this potential confound, the contralateral noninfused hippocampus served as a control in each rat.
Aberrant mossy fiber sprouting was measured by drawing contours around the entire granule cell layer (g) + molecular layer (m) (magenta line) and the Timm-positive part (cyan line) (A). h, Hilus; CA3, CA3 pyramidal cell layer. B, Isolated outlines with Timm-positive contour filled. Areas were recorded, and the percentage of the entire granule cell layer + molecular layer that was Timm positive was calculated.
To measure activation of the mTOR pathway, ribosomal protein S6 was evaluated, which is a downstream phosphorylation target of the mTOR signaling pathway (Chung et al., 1992). In an experiment to measure mTOR activity after status epilepticus, male Sprague Dawley rats (41 d old) were vehicle or pilocarpine treated, as described above. Tissue was collected for Western blot analysis 24 h or 7 d later. Control groups consisted of vehicle-treated rats (n = 5) and pilocarpine-treated rats that did not develop status epilepticus (n = 6). Control tissue was collected 24 h after treatment. Tissue was collected from pilocarpine-treated rats that experienced status epilepticus 24 h (n = 6) or 7 d previously (n = 6). To collect tissue, rats were killed by urethane overdose (2 g/kg, i.p.) and decapitated. Brains were quickly removed from the skull, and both hippocampi were immediately isolated and frozen in liquid nitrogen and preserved at −80°C. Tissue samples (25 mg) were homogenized at 4°C in 150 μl of lysis buffer consisting of 62.5 mm Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mm DTT, and 0.01% bromophenol blue. Samples were sonicated for 15 s, and aliquots were stored at −80°C. Expression levels of total ribosomal protein S6 and phosphorylated ribosomal protein S6 were measured by Western blotting. Isolated protein samples were heated to 95–100°C for 5 min, chilled on ice, and centrifuged. Isolated proteins (3 μl, ∼35 μg) were diluted and loaded on 4–20% SDS-polyacrylamide gels and electrophoresed at 20 mA/gel for 110 min, before being transferred onto pure nitrocellulose membranes (Schleicher and Schuell Bioscience) at 200 mA for 108 min. Blotted nitrocellulose membranes were blocked with freshly prepared Tris-buffered saline with 0.1% Tween 20 (TBS/T) containing 5% nonfat milk for 1 h, then incubated in rabbit anti-phospho-ribosomal protein S6 (Ser235/236) serum (1:900; Cell Signaling Technology) in TBS/T containing 5% bovine serum albumin overnight with agitation at 4°C. After a wash, nitrocellulose membranes incubated in anti-rabbit horseradish peroxidase-linked conjugate (1:5000; GE Healthcare) in TBS/T for 50 min at room temperature with agitation. Nitrocellulose membranes then were washed with TBS/T. ECL Western blotting detection reagents and autoradiography film (GE Healthcare) were used to detect bands. After completing analysis of phospho-S6 bands, blotted nitrocellulose membranes were washed in Restore Western blot stripping buffer (Pierce Biotechnology) for 15 min at room temperature and incubated with anti-total ribosomal protein S6 serum (1:2500; 5G10, Cell Signaling Technology) using methods described above. Phosphorylated S6 and total S6 levels were quantified by densitometry using NIH Image software. Ratiometric data for each animal, consisting of duplicate sample tubes, were averaged together.
In an experiment to test the effect of rapamycin infusion on mTOR activity in the hippocampus close to the infusion site, six naive male Sprague Dawley rats (35 d old) were implanted for rapamycin infusion as described above. After 14 d of infusion, rats were killed by urethane overdose