Ceruloplasmin knock-out (CpKO) mice were generated as described previously (Harris, 1999); breeding pairs were a gift from Z.L. Harris. Wild type(WT) and CpKO, background C57BL/6 lines were maintained according to National Institutes of Health guidelines. All animal procedures were approved by the Animal Care and Use Committee of the National Institute on Aging Intramural Research Program. All mice were male and three months of age when behavioral testing was initiated.
Twelve WT and 12 CpKO three month-old male mice were each evaluated in a series of behavioral tests performed in the following order: rotorod, open field, novel object recognition, water maze, elevated plus maze and fear conditioning. Approximately 24 hours of rest between behavioral tests was provided, except 48 hours elapsed between the end of water maze testing and beginning of elevated maze testing.
Separate groups of 12 WT and 12 CpKO mice were tested in the tail suspension test and 24 hours later in the forced swim test. On the day before behavioral testing, blood was taken from these animals for corticosterone measurements.
Hippocampi from a group of mice that had not been behaviorally tested (6 WT and 6 CpKO) were used for HPLC analysis and atomic absorption measures. Brains tissue from an additional 10 WT and 10 CpKO mice that had not been tested were analyzed by atomic absorption to gain statistical significance.
RNA transcripts from the hippocampi of a separate cohort of 5 mice for each group (WT and CpKO) were analyzed (24 hours prior to hippocampal harvest, blood was taken from these animals for corticosterone measurements and those data were combined with data from blood samples from 12 other mice per group. Thus, the final group numbers for corticosterone measurements were 17 WT mice and 17 CpKO mice.
Graphs represent group means with error bars representing the standard error of the means. One-way repeated ANOVA was used for fear conditioning measures of the training period and contextual memory. All other statistical comparisons were made using student t-test assuming equal variance in which p-values and critical ratios were calculated from a two-tail analysis.
Open Field Test
Open field testing was performed using the MEDOFA-MS system (Med Associates). Mice (n= 12/group) were placed in the center of an open field, and exploration was assessed for 15 min. Cages were cleaned with ethanol immediately after testing each mouse. The dimensions of the arena were 40 cm × 40 cm, of which the outer 10 cm were considered the peripheral zone and the inner 30 cm were considered the central zone.
Rotarod testing was performed using the ENV-577M system (Med Associates). Rotarod acceleration was set to 2–20 rpm. Mice (n= 12/group) were placed on the Rotarod for three trials of 5 min each with a 15-min rest between trials. The apparatus was routinely cleaned with water and ethanol following each session. The number of falls (maximum of 10 falls) within a 5 min time period, time of the first fall, and the length of time spent on the rod were recorded. After 10 falls the test was terminated and the mouse was returned to its home cage.
Morris Water Maze
To test reference memory, mice (n= 12/group) were trained in the water maze as described previously (Stranahan et al., 2008
) for 9 days, with four trials per day. The visual cues were black and white only to reduce the possible effects of color discrimination capabilities between strains. The platform was hidden 0.5 cm below the surface of the water, made opaque with white nontoxic paint, at a temperature of 27 °C ± 0.5 °C. The platform location was constant, and the starting points were changed every trial to avoid track memorizing. When trials ended, either when the mouse had found the platform or when 60 s had passed, mice were allowed to rest on the platform for 10 s. To test retention of the task, the platform was removed, and mice were given probe trials at 4 h after the last trial on the 9th
day and 24 hours later on the 10th
Novel Object Recognition
Testing took place in 25-cm ×25-cm opaque-walled cages. For object familiarization, mice (n= 12/group) were allowed to explore their cage in the presence of two identical objects. Mice were then returned to their home cages for 2 h, followed by a 30-min exposure to one novel object and one familiar object. Mice had no observed baseline preference to the different objects. Object preferences were automatically analyzed using the ANYmaze video tracking software. Mice that spent less than 1 min total time exploring the objects were eliminated from the data analysis. An object preference index was determined by calculating the time spent near the novel object divided by the cumulative time spent with both familiar and novel objects. Cages were routinely cleaned with ethanol following testing/habituating of mice.
In the training session, mice (n= 12/group) were placed in a contextual conditioning chamber (model MEDVFC-NIR-M; Med Associates) and allowed to explore the chamber for 2 min. At the end of 2 min, mice were subjected to three sessions of audio tone (CS, conditioned stimulus) and foot shock (US, unconditioned stimulus). Audio tone (5 kHz, 70 dB) was on for 30 s, followed immediately by a 0.5-mA, 2-s foot shock from the metal grid floor. Thirty seconds separated each session. Foot shock intensity was determined in a preliminary test on a separate cohort of animals for the minimal applicable intensity that elicited a response. On the following day, in the contextual fear session, mice were tested by being returned to the conditioning chamber for 5 min without any shock. The percentage of time freezing was recorded and used as an index of contextual memory. In the cued conditioning test (conducted 3 h following contextual conditioning) mice were tested by being returned to the chamber but in a different context. Mice were allowed to explore the chamber for 5 min without any audio tone. Following this, five audio tones were sounded for 30 s each. The percentage of time freezing until and after the audio tones was recorded and used as an index of cued memory. The test apparatus was cleaned with ethanol after testing each mouse.
Elevated Plus Maze
The apparatus consisted of a plus-shaped maze elevated 60 cm from the floor and comprising two opposite open arms, 25 cm × 5 cm each, crossed by two arms of the same dimensions enclosed by 30-cm-high walls with an open roof. In addition, a 1-cm-high edge made of clear Plexiglas surrounded the open arms to avoid falls. Each animal (n= 12/group) was placed in the middle of the maze facing the open arm. Following 5 min testing, animals were returned to their home cages. Arm preference was automatically analyzed using the ANYmaze video tracking software.
Forced Swim Test
Mice (n= 12/group) were tested for 5 min in a water-filled cylinder (50 cm depth × 20 cm diameter, with water level at a 40 cm height to prevent the mice from reaching the bottom with their tails). Water temperature was 27 °C ± 0.5 °C and was replaced between tests of different mice. Freezing periods of the mice were determined when mice were immobile with all four limbs.
Tail Suspension Test
Mice (n= 12/group) were tested for 6 min during which they were held by their tail. All test sessions were recorded in the dark using an infrared camera under infrared light and were subsequently analyzed. Freezing periods of the mice were defined as the time period during which the mouse hung without attempting to move.
Brain iron analysis
Brain regions (n= 16/group) were wet digested in 0.2% ultra-pure nitric acid using standard procedures and analyzed for iron concentration by atomic absorption spectrometry (Perkin Elmer AAnalyst 600, Perkin Elmer, Norwalk, CT) (Pinero et al., 2000). Standards were prepared by diluting a Perkin Elmer iron standard (PE#N9300126) in 0.2% ultra-pure nitric acid, and blanks were prepared with digesting and diluting reagents to control for possible contamination. All standard curves exceeded r > 0.99.
HPLC Analysis of Monoamines
Brain regions (n= 6/group) obtained from each animal were weighed, ultrasonicated in 0.1M perchloric acid, and stored at –70°C until extraction. The tissues obtained from each animal were homogenized in 0.1 M perchloric acid and centrifuged at 25,000 g for 12 min. Norepinephrine (NE), serotonin (5HT) and 5-hydroxyindol acetic acid (5HIAA) contents in supernatants of the brain structures were measured by HPLC with electrochemical detection. The analytical column was a SunFire C18 5 μm (4.6 × 150.0 mm) from Waters. The mobile phase was 0.01 M sodium dihydrogenphosphate, 0.01 M citric acid, 1.2 mM sodium EDTA, 1.2 mM sodium 1-heptane sulfonic acid, 10% methanol, pH 3.5; the flow rate was 1.0 ml/min and the column temperature was 34°C. The installation consisted of a Waters 717 Plus automated injection system, a Waters 1525 Binary pump, and a ESA Coulochem III detector. Waters Breeze system was used for analysis. Concentrations of NE, 5HT and 5HIAA were calculated as pg/mg of tissue weight
Thirty microliters of blood was taken via tail bleeds in the morning from naïve 3 month old mice (n= 17/group) that had not undergone behavior testing. The blood was spun at 2,500 g for 10 min and plasma was separated. Plasma was analyzed using a CORT RIA kit (MP Biomedicals, USA).
Single hippocampi (n= 5/group) were homogenized in 300 μl of a modified Ripa buffer. Homogenates were spun at 20,000 g at 4°C. Supernatants were separated and protein concentrations were determined by Bradford assay. Samples were diluted to 2 mg/mL and 50 μl was loaded per well and analyzed using the quantikine BDNF ELISA kit (R&D Systems, USA)
RNA extraction and real-time PCR
RNA from the hippocampus (n= 5/group) was isolated using Trizol (Invitrogen) and purified with an RNA Micro Kit (Qiagen, Valencia, CA). Following treatment with DNase I, RNA was quantified and equal amounts were retro-transcribed using the SuperScript First Strand Synthesis System (Invitrogen Life Technologies). Real-time PCR analysis was performed with a PTC 200 Pelthier Thermo Cycler and Chromo 4 Fluorescent Detector (BioRad, Hercules,CA), and Sybr® Green PCR Master Mix according to the manufacturer's instructions (Applied Biosystems, Foster City, CA). Each reaction included 3 μl of diluted (1:4) cDNA and was performed in triplicate. PCR was performed under the following conditions: 10 min at 95 °C, followed by 40 cycles of 30 s at 95 °C, 30 s at 60 °C and 1 min at 72°C. The comparative Ct method was used to determine the normalized changes of the target gene relative to a calibrator reference. The primers used in this study were as follows: mBDNF1 (NM_007540), 5′- GCT TTG CGG ATA TTG CGA AGG GTT -3′ and 5′- ACC TGG TGG AAC ATT GTG GCT TTG -3′; mBDNF2 (NM_001048139), 5’- TGA AGT TGG CTT CCT AGC GGT GTA -3’ and 5’- TGG TGG AAC TTC TTT GCG GCT TAC -3’; mBDNF3 (NM_001048141) 5’- CCA GAG CAG CTG CCT TGA TGT TTA -3’ and 5’- CCG CCT TCA TGC AAC CGA AGT AT -3’; mBDNF4 (NM_001048142), 5’- TGA CAA CAA TGT GAC TCC ACT GCC -3’ and 5’- ATG GTC ATC ACT CTT CTC ACC TGG -3’; mTrkB long (NM_001025074), 5’- TTC AGC TGC TGT TGC TGC TTC T -3’ and 5’- AAC CGC TAA ACC GGC ACG AAT ATC -3’;mTrkB short (NM_008745), 5’- TCC TGC TCA AGT TGG CGA GAC ATT -3’ and 5’- ATA GGC AAC AGC AGT CCC AGA GTT -3’; mHPRT (NM_013556), 5’- CCT GCT GGA TTA CAT TAA AGC ACT G-3’ and 5’- CCT GAA GTA CTC ATT ATA GTC AAG G-3’.