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Conceived and designed the experiments: TLH MAA LMGS GH. Performed the experiments: JSA MOD GH. Analyzed the data: JSA MOD MAA GH. Wrote the paper: TLH MAA JSA.
Mitochondrial uncoupling protein 2 (UCP2) is induced by cellular stress and is involved in regulation of fuel utilization, mitochondrial bioenergetics, cell proliferation, neuroprotection and synaptogenesis in the adult brain. Here we show that natural birth in mice triggers UCP2 expression in hippocampal neurons. Chemical inhibition or genetic ablation of UCP2 lead to diminished neuronal number and size, dendritic growth and synaptogenezis in vitro and impaired complex behaviors in the adult. These data reveal a critical role for Ucp2 expression in the development of hippocampal neurons and circuits and hippocampus-related adult behaviors.
The perinatal environment represents a critical period in brain development that determines the adult architecture of the central nervous system and related functions. In vitro approaches to study aspects of neuronal differentiation and synaptogenesis have long been used to gain a better understanding of the developmental processes of neuronal circuits. However, practical aspects of the successful maintenance of primary neuronal cultures do not represent close mimicking of the in vivo environment of neurons during the perinatal period. Specifically, the nutrient composition of culture media is vastly different from that provided by the placenta, clostrum and breast milk, the main source of fuel for developing neurons perinatally. An important characteristic of breast milk, in contrast to placental blood support, is its high content of long chain fatty acids besides glucose . We have identified mitochondrial uncoupling protein 2 (Ucp2) as a critical determinant of fatty acid utilization by adult neurons . Ucp2 promotes free radical scavenging –, which is critical for enabling fatty acid beta oxidation in neurons . This mechanism is also critical for adult synaptogenesis . Ucp2 is also implicated in protection of adult  as well as developing neurons in a febrile seizure model in rats at a time of breastfeeding . In the present study, we sought to determine whether Ucp2 induction occurs in the hippocampus perinatally, and if so, whether Ucp2-associated cellular mechanisms are involved in the development of neuronal circuits in vitro with implications for adult behavior.
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.
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.
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 13 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′. Ngn3 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.
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 (12000)  and rabbit anti-aralar antibody (loading control, 11000; a gift from doctor Araceli del Arco) and then with horseradish peroxidase-conjugated goat anti-rabbit and goat anti-mouse secondary antibodies (110000; 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.
The following primary antibodies were used: chicken anti-βIII tubulin (11000; Abcam, Cambridge, UK) and mouse anti-synaptophysin I (1500; 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 (1500) and goat anti-mouse-Cy3 (11000), 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 (120; Abcam, Cambridge, UK) as axonal marker and mouse anti-MAP2 (1500; Sigma-Aldrich) as dendrite marker were used. Secondary antibodies: goat anti-rabbit-Alexa488 (1500) and goat anti-mouse-Cy3 (11000), 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.
We analyzed Ucp2 expression in the hippocampus of mice delivered with Caesarian section (CS), vaginal birth (VB) and in various postnatal ages from animals born naturally. Ucp2 mRNA levels were induced in newborns by vaginal birth compared to age-matched mice that were delivered with Cesarean section (Fig. 1a). Subsequently, Ucp2 mRNA levels began to decrease and reached levels below the fetal ones. Ucp2 related genes involved in mitochondrial biogenesis, Nrf1 and Tfam, showed a similar expression profile (Fig. 1a). Differences in the level of the expression of these latter genes were observed between Ucp2 KO mice and their wild type littermates in each stage of development (Fig. 1b). Nrf1 mRNA expression level was decreased in the Ucp2 KO mice compared to wild type mice at E18 delivered with Caesarian section; Tfam mRNA expression level was smaller in the Ucp2 KO mice than in wild type mice at all developmental stages studied. The expression of Ngn3 mRNA, a proneural transcription factor related with hippocampal neuron development  was decreased in Ucp2 KO mice compared to wild type mice at P10 and in adults.
We next used cultured hippocampal neurons from day E18 that show a transition through a sequence of five morphological stages . The level of Ucp2 mRNA was the highest at stage 1 (Fig. 1c), which corresponds to in vivo stage of birth. Levels of Ucp2 mRNA decreased at stages of neuron differentiation (2 and 3) and showed a small increase at stage 4, when the dendrites have already been defined and neurons start to establish its synaptic contacts. Nrf1 and Tfam mRNA levels showed shifts in expression that was not always similar to changes in Ucp2 (Fig. 1c). Thus, in vitro culturing of neurons taken from mice that were surgically delivered has resemblance to the mimics the effect of vaginal delivery on Ucp2 expression.
We observed a significantly higher level of UCP2 protein expression at the day of delivery in animals that were born via VB compared to those with CS (Fig. 2). In naturally born mice, UCP2 protein remained elevated early post-nataly (P10) as well as in adulthood (Fig. 2).
To test the effect of Ucp2 induction on neuronal differentiation, cultures first were treated with the Ucp2 inhibitor, genipin  (Fig. 3). We have extensively analyzed the effect of genipin on mitochoindrial functions and how they relate to UCP2's effect . In those studies, we confirmed genipin's brain effects but we also showed that genipin has a more broad action on mitochondrial metabolism . Thus, we concluded that while genipin is not UCP2-specific, it does have an overall effect on mitochoindrial and cellular function that is consistent with an effect that opposes UCP2 action. Between 0.5 and 4 DIV, the number of neurons was significantly decreased by genipin treatment compared to control cultures (Fig. 3). Furthermore, genipin caused a decrease of the neuronal soma size, the number of primary neurites and dendrites and the number of synaptophysin positive presynaptic clusters on the soma and dendrites (Fig. 4). Next we analyzed, cultures from Ucp2 KO mice and their wild type littermates. Similar to the effect of chemical inhibition of UCP2, Ucp2 KO cultures showed decreased neuronal soma size, decreased number of primary neurites and decreased number of synaptophysin positive terminals compared to the cultures from their wild type littermates (Fig. 4).
Across a test of unconditioned response to potential threat, ucp2−/− mice displayed significantly more thigmotaxis, fewer trips to the center of the testing arena, shorter distance travelled and a slower rate of locomotion. (Fig. 5a–e).
WT, N=12; KO N=10. In this measure of exploration stress, ucp2−/− mice travelled significantly less distance that WT (KO distance, 4.9±0.8 m; WT distance,10±1.0 m; t(22)=4.0, p<0.001). As compared to WT mice, ucp2−/− made significantly fewer trips to the center of the arena (KO trips to center=2.1±0.80; WT trips to center=13.08±1.54, t(16)=6.34, p<0.0001). Time spent in contact with the walls of the apparatus was also significantly different across genotype; ucp2−/− mice displayed high levels of thigmotaxis relative to wild type animals (KO contact with wall=419.7±23 s; WT contact with wall=281.4±22.51 s, t(19)=4.3, p<0.001). As previously reported, ucp2−/− mice were significantly slower than age-matched WT controls (KO average speed m/s=0.017±0.003; WT average speed m/s=0.034±0.002, t(22)=4.8, p<0.0001).
The distance traveled in the novel arm, a proxy for exploratory behavior related to recognition of novelty differed significantly (KO travel in novel arm 2.38±0.24 m; WT travel in novel arm 3.42±0.37 m, t(11)=2.36, p<0. 04). Time spent in the novel arm also differed (KO time in novel arm 58.34±8.60 s; WT time in novel arm 95.08±10.14 s, t(12)=2.76, p<0. 02).
In adult mice, under conditions of synaptogenesis, such as triggered by voluntary exercise, the absence of Ucp2 not only blocks synaptogenesis, but decreases spine synapse number on granule cells as well as on CA1 pyramidal neurons . The role of UCP2 in promotion of scavenging of reactive oxygen species (ROS) in neurons , ,  is a likely contributor for the promotion of dendritic growth and synaptogenezis: ROS were found to be a critical negative regulator of hippocampal circuit development  and, they are important contributors to neuronal lipid metabolism  a process important for membrane fluidity.
The induction of Ucp2 mRNA by natural birth is consistent with previous findings that correlated the beneficial effects of Ucp2 and high fat content of breast milk with protection of febrile seizures in early postnatal animals . It is reasonable to suggest that Ucp2 mRNA induction may be associated with hypoxia/ischemia that accompanies vaginal birth. An intracellular mechanism that is known to be activated by hypoxia/ischemia is AMPK , which is an upstream inducer of Ucp2 induction in neurons . AMPK activation suppresses acetyl CoA carboxylase (ACC) activity eliminating the inhibitory effect of malonyl-CoA on carnitine palmitoyl transferase 1 (CPT1) activity. CPT1 activation enhances long chain fatty acid oxidation, which leads to generation of reactive oxygen species (ROS). ROS together with long chain fatty acid availability promotes Ucp2 transcription and activity , . Ucp2 activity neutralizes ROS – allowing continuous CPT1-promoted fatty acid oxidation and transcription of genes promoting mitochondrial proliferation (such as Nrf1 and Tfam as described in the present study) enabling continuous support of the bioenergetics needs of sustained neuronal firing and synaptic plasticity . This intracellular signaling should be supportive of neuronal activation and decreased vulnerability of neurons to cellular stress in any region where they are activated. Indeed, mice with enhanced Ucp2-expression in the hippocampus, substantia nigra, or striatum, resisted to neurodegeneration in models of epilepsy , Parkinson's disease , , , and global ischemia .
The current data suggests that the induction of Ucp2 by birth -associated physiological stress enables metabolic adaptation to a switch available nutrient utilization that is critical for proper survival and development of hippocampal and other brain neurons. Whether impaired Ucp2 induction by non-natural birth or by chemical interference could have long lasting effects on the functioning of the brain is an intriguing and potentially clinically relevant question. Our observation of adult UCP2 KO animals in open field and Y maze tests argue that long lasting effects of impaired UCP2 activity during development may in fact affect complex adult behaviors.
We thank Maria Garcia-Mauriño and Marya Shanabrough for technical assistance.
The authors acknowledge support from the NIH Director's Pioneer Award (DP1OD006850), NIH grants DK080000, DK 060711 and ADA 7-08-MN-25 to T.L.H; the Ministerio de Ciencia e Innovación, Spain (BFU2008-02950-C03-01) and from Comunidad de Madrid (CCG08-CSIC/SAL-3617). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.