Material and Methods

Animals and drug administration

Male Sprague-Dawley rats (Charles River Laboratories) weighing 260-380 g or 10 weeks old C57BL/6J (Jackson Laboratory) were used for most behavioral tests and western blot experiments, with the exception of mutant BDNF Val66Met knock-in mice that were generated as previously described (1). Heterozygous Met/Val mice were bred, and the 3 resulting littermate genotypes, Val/Val wildtype (WT), Val/Met, and Met/Met mice were used. The age of mice was 8–10 weeks for the behavioral studies. Animals were singly housed and maintained in standard conditions with a 12-h light/dark cycle and ad libitum access to food and water. Rats received a single injection of vehicle (saline) or GLYX-13 (3 mg/kg, i.v. tail vein, Naurex Inc.). For the verapamil blocking experiment, rats received a single ip injection of either verapamil (10 mg/kg, Sigma-Aldrich) or vehicle (saline) 30 min prior to injection of GLYX-13. BDNF Val66Met knock-in mice received a single injection of either GLYX-13 (0.3, 1 or 3 mg/kg, i.v. tail vein) or vehicle. Animal use and procedures were in accordance with the National Institutes of Health guidelines and approved by the Yale University Animal Care and Use Committees.

Surgical and infusion procedures

Rats were anaesthetized with 80 mg ketamine/6 mg/kg xylazine i.p., and bilateral 22-guage guide cannula were implanted at 0.5 mm above the site of infusion (+3.0 mm AP; ±1.0 mm ML; -4.0 mm DV to the bregma). Mice were anaesthetized with 100 mg/kg ketamine/10 mg/kg xylazine i.p., and bilateral 26-guage guide cannula were implanted at 0.3 mm above the site of infusion (+1.8 mm AP; ±0.4 mm ML; -2.5 mm DV to the bregma). In previous studies we have found that this anesthetic dose of ketamine does not produce an antidepressant response (2). Following 10-18 day of recovery, animals were bilaterally infused with a function-blocking anti-BDNF antibody (Chemicon; 0.5 µg/side) or normal sheep IgG (R&D System), K252a (Millopore; 25 pmol/side), NSC 23766 (Tocris; 3.76 nmol/side), Y-27632 (Tocris; 8 nmol/side) or saline or BDNF (R&D Systems; 100 ng/side) or PBS with 0.1% BSA at a rate of 0.25 µl/min for 2 min (for rats) or 0.2 µl/min for 1 min (for mice). After the behavioral studies, brains were collected and coronal sections were cut in a cryostat and cannula tip location was confirmed after Nissl staining.

Behavior studies: Forced Swim Test (FST)

The FST was carried out as previously described for rat and mouse (2,3). Each rat was subjected to a 15-min pre-swim in a Plexiglas cylinder (30 cm diameter, 65 cm height) filled with water (25 ± 1 ºC, 45 cm depth). After 24 hr, rats received drug treatments, and the next day (24 hr later), each rat was again placed in the swim cylinders for a 10 min period and videotaped. Data were analyzed in a blinded manner by scoring the total immobility time during the entire 10-min swim period. Each mouse was placed in a 4 L glass beaker (16 cm diameter, 24.5 cm height) containing water (25 ± 1 ºC, 15 cm depth) for 10 min and videotaped. The duration of immobility was scored between 2 and 6 min by an experimenter blinded to the treatment groups.

Novelty-suppressed feeding test (NSF)

The NSF was performed as previously described (2). Briefly, animals were food-deprived overnight and placed in an open field with a small amount of food in the center. The latency to feed was measured with a cut-off time of 15 min. After the NSF, home cage feeding (HCF) during a 10-min period was measured to verify motivation to feed.

Female urine sniffing test (FUST)

The FUST was carried out as previously described (4). Briefly, animals were habituated to a sterile cotton-tipped applicator placed into their home cage for 1 h. Then, each animal was exposed to a water-dipped cotton-tipped applicator for 5 min. After a 45-min interval, each animal was exposed to a cotton-tipped applicator infused with fresh urine from females of the same strain for 5 min. The time spent sniffing the cotton-tipped applicator was measured during exposure to both water and urine by a blinded experimenter. Time spent biting the cotton-tip was excluded from the analysis.

Locomotor activity test (LMA)

Animals were placed in a clean testing cage (46 cm × 23 cm × 20 cm for rats, 30 cm × 19 cm × 13 cm for mice) for 30 min, during which time the number of beam breaks was measured using the Med-PC software (Med Associates, St Albans, VT).

Primary cortical culture

Primary cortical culture was performed as previously described (5). Briefly, pregnant females were euthanized and cortices from E18 embryos were dissected. Following incubation in trypsin-EDTA (0.25%, Gibco) for 10 min, cortices were dissociated and neurons were plated at 0.6 million cells per well in 6-well polylysine-coated plates in DMEM (Gibco)/10% FBS (fetal bovine serum). For dentritic morphology, cells were plated on glass cover slips at 0.4 million cells per 6-well plate. For both 6-well and coverslips, medium was changed the following day to a serum-free medium containing neurobasal and B27 (Gibco) which was changed every 5 days. Cells were maintained at 37°C, 5% CO2, and 95% humidity. On DIV 10, medium was changed 4 hr prior to drug treatment. For blockade studies, neurons were treated with 1 μg/ml anti-BDNF antibody 20 min prior to GLYX-13 treatment and cells were collected 60 min following GLYX-13 treatment for phospho-ERK, phospho-mTOR, and phospho-p70S6K western blot analysis. For BDNF analysis, cells were treated for 60 min.

Western Blot

For crude synaptoneurosome preparations, PFC from adult rats was dissected and homogenized in a solution containing 0.32 M sucrose, 20 mM HEPES (pH 7.4), 1 mM EDTA, 1× protease inhibitor cocktail, 5 mM NaF, and 1 mM sodium vanadate. The homogenate was centrifuged for 10 min at 2,800 rpm at 4 ºC and the supernatant was centrifuged at 12,000 rpm for 10 min. After centrifugation, the supernatant (cytosolic fraction) was removed and the pellet (crude synaptosomal fraction) was resuspended by sonication in protein lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 2 mM EDTA, 1 mM NaVO3, 5 mM NaF and 1× protease inhibitor cocktail). Protein concentration was determined by BCA protein assay. For western blotting, equal amounts of protein (20 μg) for each sample were loaded into 4-20% SDS PAGE gel for electrophoresis. Polyvinylidene difluoride (PVDF) membranes with transferred proteins were blocked with 5% BSA or 5% skim milk in TBST (TBS + 0.1% Tween-20) for 1 hr and kept with primary antibodies overnight at 4ºC. The following primary antibodies were used: phospho-TrkB (Tyr706) (Santa Cruz Biotechnology sc135645, 1:200), TrkB (Cell Signaling #4603, 1:1000), PSD95 (Cell Signaling #2507, 1:1000), GluR1 (Cell Signaling #13185, 1:1000), Synapsin1 (Cell Signaling #6710, 1:1000) and phospho-PAK (Cell Signaling #2606, 1:1000). The next day, blots were washed three times in TBST and incubated with horseradish peroxidase conjugated anti-rabbit secondary antibody (1:5000 to 1:10000) for 1 h. After final three washes with TBST, bands were detected using enhanced chemiluminescence (ECL). The blots then were incubated in stripping buffer (Thermo Scientific) for 30 min at R.T. followed by three washes with TBST. The stripped blots were kept in blocking solution for 1 h and incubated with the primary antibody directed against total levels of the respective protein or GAPDH (Cell Signaling #5174, 1:1000) for loading control. Densitometric analysis of immunoreactivity for each protein was conducted using Image Lab (Bio-Rad). Immunoreactivity was normalized to saline-treated control group values for each protein.

BDNF analysis

Measurement of BDNF was performed as previously described (5). Briefly, medium containing an anti-BDNF antibody (Millipore, 1 μg/mL) was carefully collected following drug treatment and the secreted BDNF captured by the antibody was immunoprecipitated using Protein G-Sepharose beads (GE Healthcare). BDNF was detected via an ELISA assay (BDNF-ELISA E-max; Promega, WI). EIA 96-well plates (Corning, NY, USA) were coated with monoclonal antibody and incubated at 4°C for 18 hr. The plates were incubated in a block and sample buffer at room temperature for 1 hr, followed by a 2 hr incubation of BDNF samples with standards and then incubated with a polyclonal antibody for 2 hr at room temperature. After washing, the plates were incubated with a secondary anti-IgY antibody conjugated to horseradish peroxidase for 1 hr. Next the plates were incubated in peroxidase substrate and tetramethylbenzidine solution to produce the color reaction, which was stopped with 1 M hydrochloric acid. The absorbance at 450 nm was measured with an automated microplate reader and standard curves were plotted for each plate. Protein concentrations in each immunoprecipitate were measured using a BCA kit (Thermo Scientific) and values of BDNF were corrected for total amount of protein in the sample.

Immunohistochemistry

Under deep chloral hydrate anesthesia (250 mg/kg, i.p.), rats were perfused transcardially with PBS followed by 10% buffered formalin. Brains were removed, postfixed, cryoprotected and then frozen in powdered dry ice. Coronal sections (40 µm) were cut in a cryostat. Sections were incubated successively with 1% H2O2/PBST for 30 min, 10% normal goat serum (NGS)/PBST for 20 min, rabbit anti-c-Fos antibody (Santa Cruz sc-52, 1:500) in 5% NGS/PBST overnight, biotinylated goat anti-rabbit IgG (1:500, Vector Lab) in 5% NGS/PBST for 1.5 h, and avidin-biotin complex (ABC Elite Kit, Vector Laboratories) for 1 hr. The immunoreactive product was visualized with 3-3’-diaminobenzidine (DAB substrate kit, Vector Lab). The number of c-Fos-positive cells was counted in the regions (500 µm × 400 µm) near the injection sites in the mPFC from two sections per animal using the cell counter plug-in in ImageJ 1.50i software (NIH).

  1. Chen ZY, Jing D, Bath KG, Ieraci A, Khan T, Siao CJ, Herrera DG, Toth M, Yang C, McEwen BS, Hempstead BL, Lee FS (2006). Genetic variant BDNF (Val66Met) polymorphism alters anxiety-related behavior. Science 314:140-143.
  2. Li N, Lee B, Liu RJ, Banasr M, Dwyer JM, Iwata M, Li XY, Aghajanian G, Duman RS (2010). mTOR-dependent synapse formation underlies the rapid antidepressant effects of NMDA antagonists. Science 329:959-964.
  3. Ota KT, Liu RJ, Voleti B, Maldonado-Aviles JG, Duric V, Iwata M, Dutheil S, Duman C, Boikess S, Lewis DA, Stockmeier CA, DiLeone RJ, Rex C, Aghajanian GK, Duman RS (2014). REDD1 is essential for stress-induced synaptic loss and depressive behavior. Nat Med 20:531-535.
  4. Dutheil S, Ota KT, Wohleb ES, Rasmussen K, Duman RS (2016). High-Fat Diet Induced Anxiety and Anhedonia: Impact on Brain Homeostasis and Inflammation. Neuropsychopharmacology 41:1874-1887.
  5. Lepack AE, Bang E, Lee B, Dwyer JM, Duman RS (2016). Fast-acting antidepressants rapidly stimulate ERK signaling and BDNF release in primary neuronal cultures. Neuropharmacology 111:242-252.

2