CD1 mice were raised in the Cajal Institute and all procedures for handling and killing the animals used in this study were in accordance with the European Commission guidelines (86/609/CEE) and were approved by the animal care and use committee of the Cajal Institute. Vaginal birth occurs at around E18 in the CD1 mouse colony of the animal facility of the Cajal Institute. Ucp2 gene knockout and wild type littermates C57BL6 mice were generated as described previously (20) All procedures were approved by the Institutional Animal Care and Use Committee of Yale University.
Hippocampal Neuronal Cultures and Incubation Conditions
The hippocampus was dissected out from embryonic day 18 (E18) mouse embryos and dissociated to single cells after digestion with trypsin (Worthington Biochemicals, Freehold, NJ) and DNase I (Sigma-Aldrich). Neurons were plated on 6-wells plates or glass coverslips coated with poly-L-lysine (Sigma-Aldrich) at a density of 200–600 neurons/mm2, and they were cultured in Neurobasal supplemented with B-27 and GlutaMAX I (Invitrogen, Crewe, United Kingdom). Under the conditions used, our cultures were nearly devoid of glia. After the indicated time in vitro, the cultures were fixed for immunostaining (4% paraformaldehyde/4% sucrose in PBS) or harvested for real-time PCR. Parallel cultures were prepared by adding to the cultures medium 20 µM genipin (Wako Chemicals USA) before plating cells.
Quantitative real time polymerase chain reaction (real time PCR)
Total RNA was extracted from cultures at different stages of neuron development with illustra RNAspin Mini RNA isolation kit from GE Healthcare (Buckinghamshire, UK). On the other hand, hippocampi were dissected from E18, delivered with Caesarian section (CS) or vaginal birth (VB), P10 and adult mouse, disrupted and homogenized in lysis buffer and total RNA extracted with the same kit. First strand cDNA was prepared from 2 µg RNA using the RevertAid™ H Minus First Strand cDNA Synthesis Kit (MBI Fermentas, Bath, UK) according to the supplied protocol. After reverse transcription, the cDNA was diluted 1
3 and 5 µl were amplified by real-time PCR in 20 µl using SYBR Green Master Mix or TaqMan Universal PCR Master Mix (Applied Biosystems, AB, Foster City, CA) in a ABI Prism 7000 Sequence Detector (AB), with conventional AB cycling parameters (40 cycles of 95°C, 15 s, 60°C 1 min). Primer sequences were designed using Primer Express (AB) and were for Ucp2
: forward, 5′-ACAAGACCATTGCACGAGAG-3′
and reverse, 5′-ATGAGGTTGGCTTTCAGGAG-3′
; for Nrf1
: forward, 5′-CGCAGCACCTTTGGAGAA-3′
and reverse, 5′-CCCGACCTGTGGAATACTTG-3′
; for Tfam
: forward, 5′-GGAATGTGGAGCGTGCTAAAA-3′
and reverse, 5′-TGCTGGAAAAACACTTCGGAATA-3′
and Glyceraldehyde 3-phosphate dehydrogenase (Gapdh
), which was selected as control housekeeping gene, were analyzed using Assay-on-Demand gene expression products (AB). After amplification, a denaturing curve was performed to ensure the presence of unique amplification products. For visualizing and sequencing the PCR products, each mixture was electrophoresed in 2% (w/v) ethidium bromide-stained agarose gels. Then, bands were excised and cDNA was purified using the QIAquick PCR purification Kit (Qiagen, GmbH, Germany). One hundred nanograms of each sample were sequenced (Automatic Sequencing Center, CSIC, Madrid, Spain) with the corresponding forward or reverse primer. The obtained sequence was aligned with the expected sequence of each transcript obtained from the GenBank. All reactions were performed in triplicate and the quantities of target gene expression were normalized to the corresponding Gapdh
expression in test samples and plotted.
Western blot analysis of UCP2 protein expression
Lysate from hippocampal samples of animals at the time of birth with VB, CS or at postnatal day 10 (born naturally) or adulthood (born naturally) were processed for Western blot analyzes using UCP2 antisera and procedures as described in Horvath et al. 2002 
. Mouse hippocampi were homogenized in 20 mM Tris/HCl (pH 7.4), 10 mM potasium acetate, 1 mM DTT, 1 mM EDTA, 0,25% NP-40 and an anti-protease cocktail (Roche Diagnostics) and centrifuged at 700 g (10 min). The supernatant was then centrifuged at 10 000 g (15 min), and the pellets were resuspended in SDS-PAGE loading buffer.
Proteins were resolved by SDS-PAGE and transferred onto nitrocellulose membranes (Millipore). The membranes were blocked in Tris-buffered saline containing 0.1% Tween 20 and 2% ECL advance blocking reagent (Amersham) and incubated first with rabbit anti-UCP2 polyclonal antibody (1
and rabbit anti-aralar antibody (loading control, 1
1000; a gift from doctor Araceli del Arco) and then with horseradish peroxidase-conjugated goat anti-rabbit and goat anti-mouse secondary antibodies (1
10000; Jackson Immuno Research). Specific proteins were visualized with enhanced chemiluminescence detection reagent according to the manufacturer's instructions (Amersham). Densitometry and quantification of the bands were carried out using the Quantity One software (Bio-Rad). Statistical analysis of the data was performed using an unpaired t-test.
Immunocytochemistry, image acquisition and morphometric analysis
The following primary antibodies were used: chicken anti-βIII tubulin (1
1000; Abcam, Cambridge, UK) and mouse anti-synaptophysin I (1
500; Progen, Heidelberg, Germany). To verify that the labeling was caused specifically by the primary antibodies, it was either omitted or replaced by similarly diluted normal serum from the same species. Secondary antibodies were donkey anti-chicken-FITC (1
500) and goat anti-mouse-Cy3 (1
1000), both from Jackson Immuno Research (West Grove, PA). For the evaluation of soma size, dendritic morphology and presynaptic terminal identification in dissociated cell cultures, labeled neurons were visualized by standard epifluorescence under a 40× oil objective under a Leica microscope. Images were captured with a Leica digital camera controlled by the Leica software (Leica, Heidelberg, Germany). Somas size was evaluated using ImageJ 1.38 (NIH). Primary dendrite number i.e., the number of dendrites associated with the soma, and terminal counts were performed manually. A circular region of interest (ROI) with a diameter of 100 µm was projected onto the βIII- Tubulin labeled neuron, its center roughly coinciding with the center of the soma. Synaptic terminals contacting somata or dendrites were counted within the circular ROI.
To determine the number of neurons in the cultures, at least fifteen culture fields of 600 µm2 were photographed per stage using a reverse microscope equipped with phase contrast, at 1.5 hours after seeding and at the time corresponding to different stage of neuron development. The number of cell was counted manually.
To identify neuronal cells, rabbit anti-Tau (1
20; Abcam, Cambridge, UK) as axonal marker and mouse anti-MAP2 (1
500; Sigma-Aldrich) as dendrite marker were used. Secondary antibodies: goat anti-rabbit-Alexa488 (1
500) and goat anti-mouse-Cy3 (1
1000), both from Jackson Immuno Research (West Grove, PA).
The open field test apparatus was a square, polyurethane arena (36.5 cm×36.5 cm×30 cm, Plexiglas). The animal was placed in corner of the apparatus locomotion speed, distance traveled, entries into the central zone, and time spent in contact with the outer walls, were recorded for 5 minutes. Behavioral testing took place from 1000 to 1400 h (i.e. in the light phase of the light-dark cycle). The apparatus was cleaned with 10% ethanol after each animal exposure. ANY-Maze Software™ (Stoelting Company, Wood Dale, IL) was used to record and analyze behavioral data.
Spatial memory was assessed using the two-trial Y-maze task. A single Y-maze was made of black Plexiglas and consisted of three arms with an angle of 120° between each of the two arms. Each arm was 8 cm×30 cm×15 cm (width×length×height). The three arms were randomly designated: start arm, in which the mouse started to explore (always open), novel arm, in which the mouse started to explore (always open), novel arm, which was blocked during the first trial but open during the second trial.
The maze was placed on a flat surface within the behavioral testing room. Proximal visual cues (pictures within the arms of the apparatus) and distal visual cues (the configuration of the room, curtain, wall art) remained constant throughout testing. The floor of the maze was covered with white chip bedding. Between each trial the apparatus was cleaned with 10% ethanol and new bedding was added. Behavioral testing took place from 1000 to 1400 h (i.e. in the light phase of the light-dark cycle).
The Y-maze test consisted of two trials separated by an inter-trial interval (ITI) of 60 minutes to assess spatial memory. The first trial had a five-minute duration and allowed the mouse to freely explore only two arms (start arm and other arm) while the third arm was blocked. After a 60 min ITI, the second trial also of five minutes duration was conducted during which all three arms were accessible and novelty vs. familiarity was compared in all three arms. ANY-Maze Software™ (Stoelting Company, Wood Dale, IL) was used to record and analyze behavioral data.
All data are expressed as the mean + SEM or mean ± SEM from at least 3 experiments. Data was analyzed using the Graph Pad Prism 5.0 program (GraphPad Software, Inc., San Diego, CA). The means between two samples were analyzed by student t-test and between more than two samples by one-way ANOVA followed by Bonferroni post hoc. Significance was taken at p<0.05.