Animals

Two strains of mice were utilized in the current experiments, BS and ICR. BS male mice were used for experiment 1 (n = 60; from Taconic Farms, NY, USA), whereas ICR male mice were used for experiment 2 (n = 30; Harlan USA, Indianapolis, IN, USA) and experiment 3 (n = 30; Harlan Israel, Jerusalem, Israel). Mice were 8–9 weeks old at the start of treatment. Mice were singly housed in a colony room with 12:12 h light/dark cycle and with free access to liquids (see below) and to standard rodent food. All experimental procedures were conducted during the light phase of the light/dark cycle, followed NIH guidelines for care and use of laboratory animals, and were approved by the University of Minnesota Institutional Animal Care and Use Committee (IACUC) (protocol # 0812A55003) or by the Tel Aviv-Yaffo Academic College IACUC (protocol # mta-2011-05-3).

Drugs

Trehalose 1 or 2 % (The Endowment for Medical Research, Houston, TX, USA) was dissolved in water and presented to the mice as a replacement for their water bottles. Trehalose solution was available to the mice for 3 weeks prior to the start of the behavioral experiments and throughout the experimental days. Similar concentrations of maltose were used as control solution as well as a group receiving regular drinking water. Doses and mode of administration of trehalose were selected based on behavioral effects in models for neurological diseases (Yang and Yu 2009; Rodriguez-Navarro et al. 2010). Based on the results of experiment 1 (in BS mice), only the 2 % dose was used in the later experiments.

Maltose 1 or 2 % (Sigma, St. Lewis, MO) was dissolved in water and presented to mice similarly to trehalose (as described above).

Amphetamine (Sigma, St. Lewis, MO) was dissolved in saline and injected acutely at a 1 mg/kg dose, at 10 ml/kg volume. Amphetamine was injected IP immediately prior to the start of the amphetamine-induced hyperactivity experiment. This dose of amphetamine was repeatedly demonstrated to induce hyperactivity in these mice strains (Gould et al. 2007b; Flaisher-Grinberg and Einat 2010).

Procedure

For all experiments, mice were singly housed and administered with trehalose, maltose, or water for 3 weeks prior to the start of behavioral experiments. Consumption of fluids as well as weight of the mice was measured twice a week. Behavioral tests were conducted serially, during the light phase of the light/dark cycle, and included spontaneous activity, forced swim test, and amphetamine-induced hyperactivity.

Equipment and behavioral testing

Spontaneous activity

Spontaneous activity was tested differently in experiments 1 and 2 (US laboratory; BS mice and first experiment of ICR mice) and in experiment 3 (Israeli laboratory, experiment with ICR mice). For experiments 1 and 2, mice were singly tested in a 50 × 25 × 20 cm transparent plastic automated activity monitors (Opto3, Columbus Instruments, Columbus, OH, USA). Infrared beam crossings were recorded for 60 min in 10-min intervals, and total ambulatory activity was calculated across the entire session. For experiment 3, mice were singly tested in 40 × 40 × 35 cm transparent plastic cages located under a video camera interfaced with a computer. Behavior during a 30-min session was recorded and analyzed using the Tracker program (BioBserve, Bonn, Germany) for distance traveled and for the distribution of activity in the box. For both labs, mice were returned to their home cages and the boxes were wiped clean with a 10 % alcohol solution at the end of the session.

Forced swim test

Mice were placed for a 6-min session in a vertical cylindrical plastic container (35 cm tall × 20 cm diameter), filled to a depth of 15 ± 1 cm with tap water at 22 ± 1 °C. This depth was sufficient to ensure that mice could not escape or touch the floor of the container. At the end of the session, mice were removed from the water, dried with a paper towel, and placed back in their home cage. Water in the container was changed after each session. Sessions were digitally recorded from the side, and the last 4 min of each session were scored for immobility time (total immobility or minimal movements necessary to keep head above water) using an automated acquisition and analysis software (forced swim test (FST), Biobserve, Bonn, Germany).

Amphetamine-induced hyperactivity

Mice were administered with amphetamine or vehicle and immediately placed in either activity monitors for a 60-min session (experiments 1 and 2) or for a 30-min session in the plastic cages as described above for the spontaneous activity test (experiment 3), where activity was recorded and analyzed across the entire session as described above for the spontaneous activity test. At the end of the session, mice were returned to their home cages and the boxes were wiped clean with a 10 % alcohol solution.

Biochemistry

Brains

At the end of behavioral tests for experiment 3, mice were returned to their home cages where treatment with water, maltose, or trehalose continued for additional 2 days. At that time, four mice from each group (with no amphetamine history) were randomly selected and sacrificed by cervical dislocation followed by immediate decapitation; their brains were extracted and frontal cortex were dissected and immediately frozen in −80 °C for later analysis of the autophagy markers beclin-1 and p62.

Protein extraction

Total protein was extracted by sonication for 10 s at 4 °C and 50 % power capacity (Heat System Ultasonic INC) in 150 μl lysis buffer: 50 mM Tris–HCl pH 7.5, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 1 % (v/v) β-mercaptoethanol, 1 mM PMSF, 1 μl proteases inhibitor cocktail (Sigma-Aldrich, St. Louis, MO), 1 μl phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). After centrifugation at 10,000×g for 15 min at 4 °C, the supernatant was collected and protein concentration was determined spectrophotometrically using NanoDrop 2000 (Thermo Scientific).

Western blotting

Western blot analysis was performed according to a standard protocol used in our laboratory (Shamir et al. 2005). Each sample was tested in duplicates of 12 and 24 μg per well, to verify linearity on 10 % acrylamid gel and transferred to PVDF membrane. Primary antibodies, beclin-1 (1:1,000, Cell Signaling Technology, Danvers, MA), p62 (1:1,500, Abcam, Cambridge, UK) were diluted in 1 % non-fat dry milk, 1 % BSA, 0.01 % sodium azide in Tris-buffered saline Tween 20 (TBST). Goat anti-rabbit (sc-2004, 1:10,000, Santa Cruz, Dallas, TX) was diluted in TBST.

In the search for appropriate house-keeping proteins to correct for amount of protein loaded, we revealed that protein and mRNA levels of GAPDH (Costain et al. 2012), β-actin (Behan et al. 2009), and α/β tubulins (Bennett et al. 1991; Cannell et al. 2002; Beasley et al. 2006; Behan et al. 2009; Costain et al. 2012) have been repetitively reported to be altered in bipolar patients (Beasley et al. 2006; Behan et al. 2009), following lithium treatment (Bennett et al. 1991; Cannell et al. 2002) and due to oxidative stress (Costain et al. 2012). The tubulin proteins, the building blocks of microtubules, are also involved in autophagosomal trafficking and formation (He and Klionsky 2009) and were reported to be part of the autophagosome proteomics network (Dengjel et al. 2012). Hence, we could not use these commonly utilized loading controls. To overcome this limitation, we used the p62/beclin-1 ratio as readout of autophagy level. Because decreased p62 levels and increased beclin-1 levels are interpreted as markers of increased autophagy (Klionsky et al. 2008), we interpret decreased p62/beclin-1 ratio derived from a loaded protein extract of a given sample as augmented autophagy.

Statistical analysis

Data for all measures except amphetamine-induced hyperactivity were analyzed using a one way ANOVA with treatment (water, maltose, or trehalose) as main factor. When ANOVA results were significant (p ≤ 0.05), results were further analyzed with post hoc LSD tests. For the amphetamine-induced hyperactivity test, data were analyzed using a two-way ANOVA with amphetamine (yes or no) and treatment (water, maltose, and trehalose) as main factors followed by LSD post hoc tests.

The data related to the consumption of liquids were not always ideal due to leakage from a number of bottles. Specifically, we found leakage in one of the cages every few days. Leaking bottles were immediately replaced, but even if leakage was only for 1 day or some hours, consumption data from the cage were lost. It was therefore decided to compare the consumption of liquids at the 2-week time point and not later in order to maintain a reasonable number of intact bottles to measure per group. Moreover, in experiment 3, the leakage from bottles resulted in nonhomogeneous groups and data were therefore analyzed using the nonparametric Kruskal Wallis ANOVA.