The rTg4510 mice and parental mutant tau and tTA lines were generated and maintained for this study as previously described .
A concentration of 1 mM MB in saline was infused by pump into the CA3 of the right hippocampus of rTg4510 mice. Alzet pumps (Model 1004, 100 μL, 0.11 μl/hr; Alzet Osmostic Pumps) were filled with 100 μl of 1 mM MB or with saline 0.9%. The pumps were incubated in 0.9% saline at 37°C for 48 hrs. Mice were operated on a stereotaxic apparatus (51725 D, Stoelting, Wood Dale, IL). A midsagittal incision was made to expose the cranium and a small aperture was drilled with a dental tool over the right hippocampus to the following coordinates from bregma: anterior-posterior, -2.7 mm; lateral, -2.5 mm. The osmotic pump was inserted into a subcutaneous pocket on the back of the mouse, leading the catheter to the site of cannula (Brain infusion kit 3, Alzet) placement. The cannula, with a thin layer of cyanoacrylate (Loctite 454, Alzet), was attached to the stereotaxic cannula holder (Cannula holder 51636, Stoelting) and then lowered 3 mm ventral through the midline aperture. The incision was cleaned with saline and closed with surgical sutures. After surgery, the mice were housed individually. Infusion lasted for 28 days. Six mice were administered with MB and 7 with Saline. The mice were 7 months old at the time of surgery and they were 7 months and three weeks old at the time of water maze testing.
Ten age and gender-matched rTg4510 mice and ten wildtype littermates were administered MB (Sigma) and saccharine (Acros Organics, Geel, Belgium) in their drinking water, while another ten rTg4510 mice and ten wildtype littermates received drinking water with saccharine only. A treatment of 650 mg/day in a 70 kg human equates to 9.3 mg/kg/day. Based on a 4.5 ml of water/day rate of consumption of a 30 g mouse, ad libitum, we estimated mice to receive 9.3 mg/kg/day (0.062 mg/ml). MB or vehicle administration was initiated in 12 week-old rTg4510 and wildtype mice using drinking water supplemented with 2 mM saccharine. Treatment was maintained for four months and the supplemented water was replaced three times per week. Mice were 6 months old at the time of initial behavioral assessment and they were 6.5 months old at the time of the water maze analysis. During the course of the study one mouse died of unknown circumstances and drug, tau levels and stereological analyses were not able to measured.
Determination of Methylene Blue Concentrations in the Cerebellum
Frozen brain tissue was thawed on ice and homogenized for 2 minutes at a concentration of 100 mg/ml in homogenizing solution (ACN:PBS = 9:1). A 30 μL aliquot of the brain homogenate was added to a 1.5 mL polypropylene microcentrifuge tube and spiked with 30 μL of the internal standard solution (Methylene Violet 3RAX, 250 ng/ml). Analytical grade acetonitrile was then added (90 μL) and the sample was vortexed again for 30 s. Following highspeed centrifugation at 13,200 rpm for 10 minutes at 4°C, the supernatant was transferred to a glass vial and subjected to LC-MS analysis. The LC-MS system used for these studies was a Shimadzu (Columbia, MD) series 2010EV instrument equipped with an APCI probe to minimize ion suppression. Quantification was performed using LCMSolution Version 2.05 and a set of external standards.
The open field is used as a standard test of general activity. Animals are monitored for 15 minutes in a 40 cm square open field with a video tracking software (ANY-Maze, Stoelting, Illinois), under moderate lighting. General activity levels were evaluated by measurements of horizontal and vertical activity.
This test was performed on an accelerating rotorod apparatus (Ugo Basile, Italy) with a 3 cm diameter rod starting at an initial rotation of 4 RPM accelerating to 40 RPM over 5 minutes. Mice were tested for the time spent on the rod during each of four trials with a thirty-minute inter-trial interval. Each trial was completed when the mouse fell off the rod (distance of 12 cm) onto a spring-cushioned lever.
Elevated Plus Maze
Anxiety can be assessed through the elevated plus maze (EPM). The EPM consisted of two well-lit open arms (35 cm) facing each other and two enclosed arms (30.5 cm) also facing each other. Each arm is attached to a common center platform (4.5 cm square) and elevated 40 cm off the floor. The mouse was placed in the center platform and allowed to explore for 5 min. Video tracking software measured movement in each section (ANY-Maze, Stoelting, Illinois).
Morris Water Maze
The Morris water maze (MWM) consisted of a circular pool (1.38-m diameter) filled with opaque water at room temperature with an escape platform (15 cm × 15 cm) hidden beneath the water level (3 cm). Each mouse was given four trials per day with an inter-trial interval of 1 hour for 6 consecutive days. The time to find the platform (escape latency), the total distance traveled and the swim speed of the animals was recorded. Each animal was given a maximum of 60 seconds to find the platform. During training, if the mice failed to find the platform after 60 seconds, they were placed on the platform for 30 seconds. They were then towel-dried and placed in a cage with a heating pad underneath until dry and returned to their home cage. On day 7 the mice were subject to one probe trial in which the platform was removed and each animal had 60 seconds to search the training pool for the platform.
Brain Tissue Fractionation and Western Blot Analysis
Brain tissue was homogenized as previously described . Measurements of tau levels were performed by western blot analysis.
Fixed mouse brains were cryoprotected in successive 24 hours incubations of 10%, 20%, and 30% solutions of sucrose and then sectioned as previously described [Gordon, 2002]. Stained sections were imaged using an Olympus BX51 microscope at original 40×, 100×, or 200× final magnification. For quantification, images (original 100× magnification) of cornu ammonis (CA)1, CA3, entorhinal cortex, cortex and dentate gyrus were taken using spatial orientation cues. Quantification of positive staining product was determined using Image-Pro Plus (Media Cybernetics, Silver Springs, MD). Nissl staining was done in 0.05% cresyl violet for 5 minutes followed by differentiation in acidic water until desired color. Tissue was dehydrated through a graded series of ethanol (75%, 95% and 100%). Slides were cleared in Histoclear (xylene substitute) and coverslipped with DPX. Silver stain was performed as previously described .
Neurons that were stained with cresyl violet were counted in the the cornu ammonis 1 (CA1), the cornu ammonis 2 and 3 (CA2+3), the dentate gyrus (DG), the cerebral cortex (CX) and the striatum (STR) using the optical fractionator method of stereological counting  with commercially available stereological software (StereoInvestigator, MBF Bioscience, Williston, VT). A systematic random sampling of sections throughout the left hemibrain were stained as described above and coded to ensure blinding. The regions of interests (ROI) were defined using specific landmarks within the brain to maintain consistency. A grid was placed randomly over the ROI slated for counting. At regularly predetermined positions of the grid, cells were counted within three-dimensional optical dissectors. Within each dissector, a 1 μm guard distance from the top and bottom of the section surface was excluded. Section thickness was measured regularly on all collected sections to estimate the mean section thickness for each animal after tissue processing and averaged 35.24 μm ± 0.46 μm for all sections analyzed. The total number of neurons was calculated using the equation:
where N is total neuron number, Q- is the number of neurons counted, ssf is section sampling fraction, asf is the area sampling fraction and hsf is the height sampling fraction. Tissue from one mouse in the MB-treated rTg4510 cohort was unusable for this study. Therefore, n = 8 for this group.
Tau antibodies S202/T205, MC1 and PHF-1 were provided by Dr. Peter Davies, Albert Einstein College of Medicine; total tau antibody was purchased from Stantacruz Biotech, Stantacruz, CA. Horseradish peroxidase conjugated secondary antibodies were obtained from Southern Biotech, Birmingham, AL. Glyceraldehyde-3-phosphate dehydrogenase antibody was obtained from Meridian Life Science, Saco, ME. Actin antibody was obtained from Sigma, St. Loius, MO. P-tau refers to PHF-1.
Statistical analysis comparing 2 groups was done using an unpaired, two tailed t-test. Analysis comparing more than 2 groups was done using a one-way analysis of variance (ANOVA) with the Tukey-Kramer post-hoc test. Analysis of three or more groups during different time points (water maze learning and rotorod) was done using repeated measures 2-way ANOVA with the Bonferroni post-hoc test. Statistical analysis was done in the GraphPad Prism software.