Animals

Wild-type C57BL/6J, SIRT1flox/flox (Stock No. 008041), and Emx1-ires-Cre knockin mice (Stock No. 005628) were purchased from Jackson Laboratory and maintained by breeding colonies. Animals were housed in groups of 3–5 under a 12/12-h light/dark cycle (lights on at 0700 h) with ad libitum access to food and water. Adult male and female mice were used for the experiments. All animal procedures were approved by the Institutional Animal Care and Use Committee of University of Texas Health Science Center at San Antonio and Binzhou Medical University Hospital.

Generation of conditional SIRT1 knockout mice

SIRT1flox/flox mice possess loxP sites flanking exon 4 of the SIRT1 gene, which encodes the conserved SIRT1 catalytic domain [29]. Emx1-ires-Cre knockin mice have an internal ribosome entry site (ires) and a Cre recombinase coding region inserted into the 3′ untranslated region of the mouse Emx1 gene. The Cre transgene is principally expressed in glutamatergic neurons in the hippocampus and neocortex including the mPFC [30,31,32]. SIRT1flox/flox mice and Emx1-ires-Cre mice were maintained on a C57BL/6J background. To generate mice with SIRT1 knockout in forebrain excitatory neurons, SIRT1flox/flox mice were crossed with Emx1-ires-Cre mice to produce SIRT1flox/flox, Emx1-ires-Cre (SIRT1Emx1-KO) mice. The PCR primers used for genotyping were as follows: Emx1-ires-Cre, forward-5′-CCAGCTAAACATGCTTCATCGTC-3′, reverse-5′-GGATTAACATTCTCCCACCGTCAG-3′; SIRT1, forward-5′-GGTTGACTTAGGTCTTGTCTG-3′, reverse-5′-CGTCCCTTGTAATGTTTCCC-3′.

Drugs

SRT2104 (Selleck Chemicals LLC, Houston, TX, USA) was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 5 mg/mL and diluted in phosphate-buffered saline (PBS) (8 g/L NaCl, 0.2 g/L KCl, 0.1 g/L MgCl 2 ·6H 2 O, 0.133 g/L CaCl 2 ·2H 2 O, 0.2 g/L KH 2 PO 4 , and 1.15 g/L Na 2 HPO 4 ) to a final concentration of 0.05 µg/µL for intracranial infusion.

RNA extraction and real-time PCR analysis

Adult male SIRT1flox/flox and SIRT1Emx1-KO mice were killed by decapitation and the mPFC, hippocampus, and hypothalamus were immediately dissected on ice. For the analysis of mRNA expression in the subregions of mPFC, brain tissue from adult male mice (12-week old) was sectioned in 80-μm-thick coronal sections in a cryostat (Leica Biosystems Inc., Buffalo Grove, IL). Prelimbic and infralimbic mPFC were dissected from the 80-µm-thick coronal brain sections (six sections from 1.98 mm to 1.54 mm from bregma) on ice according to the mouse brain atlas [33]. All samples were snap frozen in liquid nitrogen after dissection and stored at −80 °C until further processing for RNA extraction, as described below. Total RNA was extracted with the RNeasy Mini Kit (Qiagen, Germantown, MD). The cDNA was generated using the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher, Waltham, MA) [34] and processed for real-time PCR quantification using the QuantStudio 3 real-time PCR system (ThermoFisher, Waltham, MA). The primer sequences used for amplification of SIRT1 and the genes involved in mitochondrial biogenesis and dynamics were as follows: SIRT1 exon 4, forward-5′-GATGCTGTGAAGTTACTGCAGGAGTG-3′, reverse-5′-GAGGGTCTGGGAGGTCTGGGAAG-3′ [35]; peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), forward-5′-TGATGTGAATGACTTGGATACAGACA-3′, reverse-5′-GCTCATTGTTGTACTGGTTGGATATG-3′; mitofusin 1 (Mfn1), forward-5′-GCAGACAGCACATGGAGAGA-3′, reverse-5′-GATCCGATTCCGAGCTTCCG-3′; mitofusin 2 (Mfn2), forward-5′-TGCACCGCCATATAGAGGAAG-3′, reverse-5′-TCTGCAGTGAACTGGCAATG-3′; mitochondrial fission 1 protein (Fis1), forward-5′-CAAAGAGGAACAGCGGGACT-3′, reverse-5′-ACAGCCCTCGCACATACTTT-3′; dynamin-related protein 1 (Drp1), forward-5′-ATGCCAGCAAGTCCACAGAA-3′, reverse-5′-TGTTCTCGGGCAGACAGTTT-3′ [36]; β-tubulin, forward-5′-AGCAACATGAATGACCTGGTG-3′, reverse-5′-GCTTTCCCTAACCTGCTTGG-3′.

Behavioral procedures

Mice were weighed weekly from 3 to 9 weeks of age. Food consumption (standard chow diet) was measured for 2 consecutive days. Average daily food intake was calculated and adjusted for body weight. Adult male and female mice (8–15 weeks) were used for behavioral tests. All behavioral procedures were performed in the late light cycle except the saccharin preference test and scored by experimenters who were blind to the genotypes or treatments. Mice were subjected to multiple behavioral tests spaced at least 3 days apart to decrease possible carryover effects from previous test. The order of behavioral tests was the same for each mouse; each mouse was tested once per test.

Saccharin/sucrose preference test

Mice were habituated to drinking water from two bottles for 1 week before testing. Mice were provided with a free choice of either drinking 0.01% saccharin solution or plain water. A computerized “lickometer” connected to an operant chamber (Med Associates Inc., Fairfax, VT) equipped with two bottles was used to determine the hedonic response of mice to sweet solutions. Mice were habituated to the operant chamber and water deprived overnight. Mice were offered a choice between saccharin solution and a lickometer recorded the number of licks to each sipper tube. The preference was calculated as the number of licks to the saccharin sipper tube divided by the total number of licks to all the sipper tubes times 100. The saccharin/sucrose preference was also measured in the home cage. Mice were provided with a free choice between a bottle containing saccharin (0.01%) or sucrose (1%) solution and a water bottle. Water and saccharin/sucrose intake was measured, and the preference for saccharin or sucrose was calculated by dividing the mass of saccharin solution consumed by the total mass of fluid intake.

Female urine sniffing test

This is a non-operant test to assess sex-related reward-seeking behavior based upon interest of male rodents in pheromonal odors from estrus female urine [37]. Male mice were subjected to the following test procedure: (1) 3-min exposure to the cotton tip dipped in water; (2) a 45-min interval; (3) 3-min exposure to the cotton tip dipped in fresh urine collected from female mice in the estrus phase; (4) a 45-min interval; and (5) 3-min exposure to the cotton tip dipped in fresh urine from male mice. The duration of female urine sniffing time was scored.

Forced swim test

Mice were placed in a clear Plexiglas cylinder (25 cm high; 10 cm in diameter) filled with 24 °C water to a depth of 15 cm. A charge-coupled device (CCD) camera positioned directly above the cylinder was used to record the behavior of each mouse for 6 min. The duration of immobility in the last 4 min was measured. Immobility was defined as no movement of the limb or body except those caused by respiration [34, 38, 39].

Locomotor activity

Mice were placed in the SuperFlex Fusion open field cages (40 × 40 × 30 cm3, Omnitech Electronics Inc., Columbus, OH) and allowed to freely explore for 30 min under the illuminated conditions. The movements of mice were monitored by infrared photosensors equipped on the cage and the total distance traveled was analyzed through the Fusion software (Omnitech Electronics Inc., Columbus, OH).

Learned helplessness test

The learned helplessness test was performed in a shuttle cage divided equally into two chambers with an auto-controlled guillotine door between the two chambers (Coulbourn Instruments, Holliston, MA) as previously described [38, 40]. Mice were subjected to 200 scrambled, inescapable foot shocks (0.3 mA shock amplitude, 2-s duration, 16-s average interval) over a 1 h session for 2 consecutive days to induce learned helplessness. Escape performance was tested 24 h after the last session in the same shuttle cage. Each mouse was given 30 shuttle escape trials with 25-s maximum duration and 30-s intervals. A sound cue and the shock took place at the same time as the guillotine door opened in the first five trials. For the remaining trials, the guillotine door opened 2 s after the shock was delivered. Each trial was terminated when the mouse crossed into the non-shock compartment. The latency to escape in each trial during the test were recorded automatically by the Graphic State software (Coulbourn Instruments Inc., Holliston, MA).

Chronic unpredictable stress (CUS)

Mice (9-week old) were subjected to a variety of stressors at different times of the day for 10 days. The stressors included 2-h restraint, 15-min tail pinch, 24-h constant light, 24-h wet bedding, and 45° cage tilt; 10-min inescapable foot shocks; and 30 min elevated platform and social isolation. Stress exposure was conducted in a procedure room, and the mice exposed to the CUS procedure were singly housed. Control mice were group housed and briefly handled daily in the housing room.

Whole-cell patch-clamp recordings

Electrophysiological recordings were performed as previously described [41]. Male mice (8-week old) were anesthetized with isoflurane and brains were quickly transferred to an ice-cold solution (254 mM sucrose, 3 mM KCl, 2 mM MgCl 2 , 2 mM CaCl 2 , 1.25 mM NaH 2 PO 4 , 10 mM d-glucose, and 24 mM NaHCO 3 ). Coronal brain slices (300 μm) containing the mPFC were prepared with a Leica VT1000S vibratome (Leica Microsystems) and allowed to recover at 30 °C for at least 1 h in an oxygenated (95% O 2 /5% CO 2 ) artificial cerebrospinal fluid solution. Patch electrodes with tip resistances between 4 and 7 MΩ were filled with a potassium gluconate-based internal solution (120 mM potassium gluconate, 20 mM KCl, 2 mM MgCl 2 , 10 mM HEPES, 2 mM ATP, 0.25 mM GTP, and 0.1 mM EGTA adjusted to 7.4 and osmolarity of 295 mOsm). Pyramidal neurons in the layer V of mPFC were visualized with a 40× water-immersion lens and recorded with the Multiclamp 700 A amplifier (Molecular Devices, Sunnyvale, CA). To investigate the firing properties of these neurons, current-clamp recordings were made from a −70 mV holding current and incremental stepwise positive current injections (15 pA) for a 0.5 s duration. Spontaneous excitatory postsynaptic currents (sEPSCs) were recorded in the voltage-clamp mode with membrane potentials held at −70 mV in the presence of picrotoxin (100 μM). Tetrodotoxin (TTX, 1 μM) was added to block action potential (AP) formation and its propagation for the recording of miniature EPSCs. For neuronal excitability in response to current injections, pyramidal neurons in the prelimbic and infralimbic mPFC were recorded on brain slices from the same mice. For sEPSCs and miniature EPSCs, pyramidal neurons in the prelimbic and infralimbic mPFC were recorded from different mice.

Electron microscopic detection of mitochondria

Male mice (8-week old) were transcardially perfused with 0.1 M PBS followed by 2% paraformaldehyde (PFA) and 2% glutaraldehyde. Brains were removed and coronal brain sections were sliced at 1 mm with Leica VT1000S vibratome (Leica Microsystems, Wetzlar, Germany). Then the brain slices were modified into 1 mm3 tissue blocks containing the mPFC. The following electron microscopic procedure was performed by the Pathology Electron Microscopy Facility of the University of Texas Health Science Center at San Antonio using the standard techniques as described by others [42, 43]. Briefly, the brain tissue blocks were fixed in a phosphate-buffered solution containing 4% formaldehyde and 1% glutaraldehyde for 2 h and further fixed in 1% Zetterqvist’s buffered osmium tetroxide for 90 min at room temperature. After washing, brain tissues were dehydrated with a series of ethanol (70%, 95%, 100%, 100%), cleared twice in propylene oxide, and infiltrated with propylene oxide–resin (v/v = 1:1) followed by 100% resin buffer for 90 min. Tissues were then embedded in plastic resin. Orientation and section quality was checked with 1-μm-thick sections stained with 1% toluidine blue under a light microscope. Ultrathin sections (90 nm) were cut and collected on standard copper grids counterstained with 1% uranyl acetate for 10 min with Reynold’s lead citrate and viewed in a FEI Tecnai G2 Spirit transmission electron microscope. The images were achieved with AMT’s CCD imaging system. The sections containing a visible nucleus were randomly selected and analyzed by an experimenter blind to the genotype of mice. The total number of mitochondria contained within the cytoplasmic surface area was quantified using ImageJ. The total area of cytoplasm was measured. Mitochondrial density was estimated by dividing the total number of mitochondria by the cytoplasmic area and was expressed as the number of mitochondria per square micrometers of cytoplasm.

Separate cohorts of SIRT1flox/flox and SIRT1Emx1-KO mice were used for behavioral phenotyping, whole-cell patch-clamp recordings, electron microscopic detection of mitochondria, and real-time PCR analysis of expression of the genes involved in mitochondrial biogenesis and dynamics.

Stereotaxic surgery, microinjection, and cannulation

Stereotaxic surgery was performed under anesthesia as previously described [38, 41, 44]. For intra-mPFC viral injection, AAV5-CMV-Cre-GFP (in the following referred to as AAV-Cre-GFP) containing the genes for Cre recombinase and green fluorescent protein (GFP) and control AAV5-CMV-GFP (in the following referred to as AAV-GFP) containing the gene for GFP alone with titers >1 × 1012 vg/mL (UNC Vector Core, Chapel Hill, NC) were injected bilaterally into the mPFC (coordinates: anterior–posterior (AP) = 1.8 mm, medial–lateral (ML) = ± 0.4 mm, dorsal–ventral (DV) = −2.6 mm from the bregma) of adult male SIRT1flox/flox mice (7-week old). A total volume of 0.5 μL adeno-associated viral (AAV) vectors (per side) was delivered at a rate of 0.10 μL/min with a 33-gauge stainless steel injector connected to a UMP3 micro syringe pump (World Precision Instruments, Sarasota, FL). Behavioral tests were performed 3 weeks after AAV injection. The injection sites were verified in each animal at the end of the experiments (Fig. 1j). Mice with “missed” injections were excluded from statistical analysis.

For intra-mPFC microinjection of the SIRT1 activator SRT2104, a 26-gauge double-guide cannula (Plastics One) was implanted 1 mm above the mPFC (coordinates: AP = 1.8 mm, ML = ± 0.4 mm, DV = −1.6 mm from the bregma) of adult male C57BL/6J mice (8-week old). Following surgery, mice were individually housed and allowed to recover for 7 days before the chronic stress procedure. Microinjections were performed on freely moving mice in their home cage. On the experimental day, a bilateral injection cannula (33-gauge) connected to a 5-μL syringe was inserted into the guide cannula and extended 1 mm beyond the tip. SRT2104 (0.05 µg/µL) or vehicle (1% DMSO in PBS) were infused into the mPFC in a volume of 0.2 µL over 2 min using an infusion pump (KD Scientific Inc., Holliston, MA). Injectors were held in place for an additional 5 min after the infusion to avoid backflow. Mice were given three infusions of SRT2104 within 24 h (23, 3, and 1 h) before behavioral testing. This sub-chronic drug treatment paradigm was based upon three injections over a 24-h period for testing antidepressants that was first reported by Porsolt et al. [45]. The purpose of multiple SRT2104 injections was to induce sufficient activation of SIRT1.

For intracerebroventricular (ICV) injection of the SIRT1 activator SRT2104, a guide cannula (C315GS, Plastics One) was implanted into the lateral ventricle (coordinates: AP = −0.2 mm, ML = 1.1 mm, DV = −1.7 mm from the bregma) of adult male C57BL/6J mice (8-week old) as described previously [38]. The injection cannula (33-gauge) connected to a 5-μL syringe was inserted into the guide cannula and extended 1 mm beyond the tip. SRT2104 (0.03 µg/µL) or vehicle (0.67% DMSO in PBS) were infused into the lateral ventricle in a volume of 1.0 µL over 2 min using an infusion pump. Injectors were held in place for an additional 5 min after the infusion. Mice were given two infusions of SRT2104 within 24 h (23 and 3 h) before behavioral testing.

Immunohistochemistry

To detect protein expression of SIRT1 in the brain, immunohistochemistry was performed as described previously [46,47,48]. Briefly, adult male C57BL/6J mice (8-week old) were transcardially perfused under anesthesia through the ascending aorta using 0.1 M PBS followed by 4% PFA in PBS. The brains were removed and fixed overnight in 4% PFA and then transferred to 30% sucrose in PBS. Brains were cut into 40-μm coronal sections on a cryostat and stored in cryoprotectant (30% sucrose, 30% ethylene glycol, 1% polyvinyl pyrrolidone, 0.05 M sodium phosphate buffer) until processing for immunohistochemistry. Free floating sections were first treated with 1% hydrogen peroxide in PBS to quench the endogenous peroxidase. The tissue was then incubated in immuno blocking buffer (3% goat serum, 1% bovine serum albumin, 0.3% triton-X 100 in PBS) for 1 h at room temperature followed by rabbit anti-SIRT1 (1:1000, #07–131, EMD Millipore, Burlington, MA, USA) in blocking solution for 48 h at 4 °C. After rinsing in PBS buffer, the sections were incubated with goat anti-rabbit IgG secondary antibody conjugated to horseradish peroxidase (1:500) at room temperature for 4 h. The sections were washed in PBS and developed with the DAB Peroxidase Substrate Kit (Vector Laboratories, Burlingame, CA). Olympus BX51 microscope (Olympus Scientific Solutions America, Waltham, MA, USA) was used to visualize the immunostaining in the mPFC and capture images.

Statistical analysis

All results are presented as mean ± s.e.m. (standard error of mean). Shapiro–Wilk test and F test were used to test the normality and equal variance assumptions, respectively. For normally distributed data, two-tailed t tests were used to assess differences between two experimental groups with equal variance. For a two-sample comparison of means with unequal variances, two-tailed t tests with Welch’s correction were used. One-way analyses of variance (ANOVAs) followed by Sidak post hoc tests were used for analysis of three or more groups. For non-normally distributed data, Mann–Whitney U tests were performed to compare two groups. For analysis of three or more groups with non-normally distribution, the Kruskal-Wallis test followed by Dunn's multiple comparisons test was used. For locomotor activity, the escape latency in the learned helplessness test, and the number of APs elicited by current injections, two-way repeated-measures ANOVAs followed by Bonferroni tests were used. P < 0.05 was considered statistically significant.