Inhibitory Avoidance

Inhibitory Avoidance

The inhibitory-avoidance apparatus (Ugo Basile, Comerio, Italy) consisted of a Plexiglas cage with tilting floor, divided by a sliding door into two compartments (22×22×25 cm each). One of the compartments had white walls and was brightly illuminated by a 10-W bulb. The other compartment had black walls and was not illuminated. The tilting floor consisted of bars of stainless

steel connected to a source of scrambled shock. The procedure consisted of two sessions, acquisition and retention, that took place on 2 subsequent days (Mereu et al, 2003);(Campolongo et al, 2007). On the first day, rats were singularly placed in the illuminated compartment and, after a 10-second delay, the sliding door was opened allowing the animal to freely explore the apparatus. The time taken by the animal to completely enter into the dark compartment was measured (approach latency) and taken as an index of non-associative behavior. Once the animal was in the dark compartment, the sliding door was closed to prohibit it from escaping and a mild shock (0.6 mA) was delivered to the floor grids for 2 seconds. Then, the animal was returned to its home cage and tested for retention 24 hours later. In the retention session, the animal was placed in the lit compartment following the same procedure as in the acquisition session and the latency to re-enter (avoidance latency) the dark compartment was recorded and assumed to be a measure of memory retention. Both acquisition and retention trials lasted for a maximum observation time of 300 seconds.

During the acquisition session, the response of the animal to the electric shock was evaluated according to the procedure described by Hiramatsu and Inoue (Hiramatsu and Inoue, 1999).

The following scores were given based on the responses to the electric shock: 3=jumping, 2=vocalization, 1=inching, 0=no response.

Shock sensitivity is shown as a total score, that was the sum of each score for 15 s after foot shock during the acquisition session.

HPLC-UV analysis of isoprenoids

Cholesterol, Coenzyme Q9 (CoQ9), Coenzyme Q10 (CoQ10) and dolichols were analyzed according to the previously described protocol (Tang et al, 2001) with modifications. Briefly, two parallel runs, one for CoQ9 and CoQ10 and another for cholesterol and dolichols, were carried out on a 4.6- x 75-mm Zorbax XDB-C18 (3.5 µm) reversed-phase column (Agilent, Santa Clara, CA, USA) with a Waters dual-pump apparatus, a Waters gradient programmer, and a Waters photodiode array detector (spectrum range: 210–400 nm; Waters Corp., Milford, MA, USA). A combination of convex gradients (Waters No. 5, from 0 to 75% B during the first 20 min and linear, from 75 to 100% B during the following 10 min) was used for the elution; in the last 5 min, reequilibration back to 0% B was performed, where solvent A was methanol: water (9:1 v/v), and solvent B was methanol:propan-2-ol:hexane (2:1:1 v/v/v). The solvent flow rate was 1.5 ml/min. HPLC solvents were obtained from POCh (Gliwice, Poland). The chain length and identity of lipids were confirmed by the addition of the internal standards. CoQ6 and dolichol-23 were from the Collection of Polyprenols (Institute of Biochemistry and Biophysics, PAS, Warsaw, Poland) and stigmasterol was from Fluka-Sigma-Adrich, Poland.

Lysate, cytosol and membrane preparation from brain tissue

Lysate and membrane from brain tissue were prepared slightly modifying our previously used protocol (Segatto et al, 2011). Briefly, the brain regions of interest (amygdala, hippocampus, prefrontal cortex, dorsal and ventral striatum) were homogenized in a homogenization buffer (0.01 M Tris-HCl, 0.001 M CaCl2, 0.15 M NaCl, 0.001 M PMSF, pH 7.5) to yield the total membrane and lysate. An aliquot of homogenate was collected and used for the lysate preparation. The remaining homogenate was spun at 10,000 g for 20 min. The supernatant (S1) was centrifuged at 100,000 g for 45 min. The resulting supernatant (S2) was collected as cytosolic fraction, while the pellet (P2) was resuspended in homogenization buffer and centrifuged again at 100,000 g for 45 min. The pellet (P3), considered as the membrane fraction, was solubilized in sample buffer (0.125 M Tris-HCl -pH 6.8- containing 10% SDS, 0.001 M PMSF) and transferred into 1.5 ml tubes. The aliquot of homogenate held for lysate preparation was resuspended in sample buffer by sonication. Protein concentration was evaluated by the method of Lowry et al. (Lowry et al, 1951). All membrane and lysate samples were boiled for 3 min before loading to the SDS-Page for subsequent Western blotting analysis. The protein detection of caveolin (membrane marker) and α-tubulin (cytosolic marker) verified and confirmed a high degree of purity of the membrane fractions (Figure S1).

Rab3 “in vitro” degradation assay

Rab3 degradation assay was performed according to the protocol used by Pallottini et al., (Pallottini et al, 2004) with modifications. Briefly, cytosolic fractions of amygdala, hippocampus, prefrontal cortex, dorsal striatum and ventral striatum samples from simvastatin-treated and control rats were homogenized in ice cold 0.01 M Tris-HCL (pH 7.4), 0.150 M sucrose and incubated at 37°C. Total protein concentration was assessed through the method of Lowry et al. (Lowry et al, 1951).50 µg homogenate was used for each reaction. After each chosen time interval, the incubation was stopped and the reaction was blocked by adding an equal volume of sample buffer (as above mentioned). Then, all the samples were boiled for 3 minutes and employed for Western blot analysis.

Co-immunoprecipitation

Cytosolic fractions from amygdala, hippocampus and prefrontal cortex were immunoprecipitated as described previously with modifications (Han et al, 2002).Briefly, 500 μg of proteins were incubated with either 5μg of RhoGDI (A-20, Santa Cruz Biotechnology) antibody or 5μg of RabGDI (V-20-R, Santa Cruz Biotechnology) antibody. The cytosolic fractions were incubated together with the antibodies for 60 min at 4°C, and then 50 μl of protein A-agarose was added for 60 min at 4°C. After centrifugation (50,000 g for 15 min), the supernatant was collected (as immunodepleted fraction) and the pellet was washed with washing buffer 3 times, to eliminate non-specific binding. For each wash, the pellet was gently mixed with washing buffer, and centrifuged at 4°C.After the last centrifugation, the supernatant and the immunoprecipitated proteins were separated in 12% SDS-PAGE. Proteins were electrophoretically transferred onto nitrocellulose and then probed overnight at 4°C with anti-Rab3 or anti-RhoA antibodies.

Hippocampal neuron primary cultures and drug treatment

Primaryneuronal cell cultures were prepared from the hippocampiof 18-day-old Wistar rat embryos (Malchiodi-Albedi et al, 2001). Fetuses were removed from maternal rats anesthetized with CO2 and killed by decapitation. Hippocampi were collected in Hank’s balanced salt solution and mechanically fragmented. After dissociation, cells were seeded onto poly-L-lysine-treated dishes in MEM and 10% fetal bovine serum (FBS). After 2 h, the medium was replaced with neurobasalmedium, supplemented with B27 (NBM/B27)(Gibco, Burlington, ONT). The experiments were performed after 1 day in vitro. In order to study the involvement of HMGR inhibition on Akt activation, 1 µM simvastatin (Sigma) in DMSO, 100 µM mevalonate (Sigma), 10 µM geranylgeraniol (Institute of Biochemistry and Biophysics, Warsaw, Poland) and 10 µM hydroxyfasudil (Sigma) were used for a 24 hours treatment. Total lysate, cytosol and total membrane were obtained by slightly modifying the protocol used for lysate, cytosol and membrane preparation from brain tissue.

Western blotting analysis

Western blot method was performed by slightly modifying the protocol described by Trapani et al. (Trapani et al, 2011).Proteins (30 µg) from synaptic vesicle, cytosol, membrane and lysate samples were resolved by 12% (for Synaptophysin, Caveolin, RhoGDI, RabGDI, RhoA, Ras and Rab3), 10% (for p-Akt, t-Akt, NeuN, CREB, p-CREB and GFAP), and 7% (for LDLr and nSREBP-2) SDS-PAGE at 40 mA (constant current) for 60 min. Subsequently, the proteins were electrophoretically transferred onto nitrocellulose membrane in cold room for 120 min at 80 V. The nitrocellulose membrane was blocked at room temperature with 5% fat-free milk in Tris-buffered saline (0.138 M NaCl, 0.027 M KCl, 0.025 M Tris-HCl, and 0.05% Tween-20, pH 6.8), and probed at 4°C overnight with primary antibodies followed by incubation for 1 h with horseradish peroxidase-conjugated secondary IgG antibodies (Bio-Rad Laboratories, Milan, Italy). The nitrocellulose membrane was then stripped with Restore Western blot stripping buffer (Pierce Chemical, Rockford, IL, USA) for 15 min at room temperature and re-probed with anti-tubulin (α-tubulin DM-1A; Sigma) antibody. Bound antibodies to proteins onto nitrocellulose were visualized by using enhanced chemoluminescence detection (GE Healthcare) and exposure to Amersham Hyperfilm ECL (GE Healthcare). The images derived from Western blotting were analyzed by ImageJ (National Institutes of Health, Bethesda, MD, USA) software for Windows. All samples were normalized for protein loading by using α-tubulin (chosen as housekeeping protein) and/or Ponceau S staining. Thus, each reported value was derived from the ratio between arbitrary units obtained by the protein band and the respective tubulin or Ponceau S staining.

Immunohistochemistry

Three simvastatin-treated rats and three untreated animals were anesthetized and perfusion-fixed, as previously described (Moreno et al, 1995). Brains were dissected out, cut in halves and paraffin-embedded; serial, 5-μm-thick, sagittal brain sections from each group were deparaffinized and processed for immunohistochemistry (Fanelli et al, 2013). The following two primary antibodies were used: 1:100 rabbit polyclonal anti-pCREB (Santa Cruz Biotechnology); 1:100 mouse monoclonal anti-PSD95 (Millipore, Billerica, MA, USA). In control sections, the primary antibody was omitted. Biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA), or biotinylated goat anti-mouse IgG (Vector) were used as secondary antibodies. Immuno-complexes were visualized, and slides were dehydrated, mounted adn photographed as previously reported (Fanelli et al, 2013). Electronic images were composed in an Adobe Photoshop CS5 format.