Drd2a-eGFP (C57Bl/6) mice were generated as described previously (Gong et al., 2003). rpS6^P−/− knockin (C57Bl/6/Sv129/ICR; Ruvinsky et al., 2005) mice were generated by crossing heterozygous rpS6^P+/– mice. Each transgenic mouse was compared with wild-type littermates of the same genetic background. Male and female were used for all experiments. Animals were housed under standardized conditions with a 12 h light/dark cycle, stable temperature (22 ± 1°C), controlled humidity (55 ± 10%) and food and water ad libitum. All experiments were in accordance with the guidelines of the French Agriculture and Forestry Ministry for handling animals and were approved by the local Ethic Committee (A 34-172-41).

Mice were killed by cervical dislocation and the heads were immersed in liquid nitrogen for 4 s. The brains were then removed and the striatum, hippocampus, or frontal cortex were rapidly dissected on an ice-cooled disc. For the dorsal striatum (DS) and nucleus accumbens (Acb), the brains were sectioned. The Acb was isolated from a ~1-mm thick coronal section located between 1.94 mm and 0.86 mm anterior to bregma and the DS between 0.86 mm and 0.14 mm. Both structures were dissected on an aluminum block on ice.

The brain regions of interest were rapidly dissected on an ice-cooled disc, sonicated in 300 μl 10% sodium dodecyl sulfate (SDS) and boiled at 100°C for 10 min. In each experiment, samples from all animal groups were processed in parallel to minimize inter-assay variations. Protein quantification and western blots were performed as described (Biever et al., 2015a). The antibodies used are summarized in Table 1. For quantitative purposes, the optical density values of active phospho-specific antibodies were normalized to the detection of nonphospho-specific antibodies or to β actin values in the same sample and expressed as a percentage of control mice.

Tissue preparation and immunofluorescence were performed as described (Biever et al., 2015a). Primary antibodies used are listed in Table 1. Goat Cy3-coupled anti-rabbit (1:500, Jackson ImmunoResearch Laboratories), donkey Cy3-coupled anti-goat (1:1000, Jackson ImmunoResearch Laboratories) and goat Alexa Fluor 488-coupled anti-mouse (1:500, Invitrogen) secondary antibodies were used. Three slices per mouse were used in all immunofluorescence analyses (n = 3–4 mice/staining).

Coronal 200–250 μm slices of mouse brain were prepared in cooled artificial cerebrospinal fluid (ACSF) containing (in mM): NaCl 119, KCl 2.5, MgCl 1.3, CaCl₂ 2.5, Na₂HPO₄ 1.0, NaHCO₃ 26.2 and glucose 11, bubbled with 95% O₂ and 5% CO₂. Slices were kept at 32–34°C in a recording chamber superfused with 2.5 ml/min ACSF. Visualized whole-cell patch-clamp recording techniques were used to measure synaptic responses of putative D1R and D2R-MSNs of the striatum, identified by the presence of the eGFP of BAC transgenic mice by using a fluorescent microscope (Olympus BX50WI, fluorescent light U-RFL-T). Whole cell recordings in voltage clamp mode. The holding potential was −70 mV, and the access resistance was monitored by a hyperpolarizing step of −14 mV with each sweep, every 10 s. Experiments were discarded if the access resistance varied by more than 20%. The internal solution contained (in mM) 140 k-gluconate, 5 KCl, 10 HEPES, 0.2 EGTA, 2 MgCl₂, 4 Na₂ATP, 0.3 Na₃GTP, and 10 sodium creatine-phosphate. Currents were amplified (Multiclamp 700B, Axon Instruments), filtered at 5 kHz and digitized at 20 kHz (National Instruments Board PCI-MIO-16E4, Igor, WaveMetrics). The liquid junction potential was small (–3 mV), and therefore traces were not corrected. Experiments were carried out in the presence of picrotoxin (100 μM). Synaptic currents were evoked by stimuli (50–100 μs) at 0.1 Hz through bipolar stainless steel electrodes placed on the slice. The magnitude of HFS (100 pulses at 100 Hz repeated four times at 0.1 Hz paired with depolarization at 0 mV)-induced LTP was determined by comparing average EPSCs that were recorded 25–30 min after induction to EPSCs recorded immediately before induction. Paired-Pulse ratio (PPR) was determined with an inter-stimulation interval of 50 ms. Spontaneous EPSCs were recorded with the same conditions and frequency and amplitudes of these currents were then analyzed using the Mini Analysis software package (v.4.3, Synaptosoft). For recordings of neuronal firing as a function of current step injection, rheobase and resting membrane potential (RPM), the internal solution contained (in mM): potassium gluconate 130, MgCl₂ 4, Na₂ ATP 3.4, Na₃ GTP 0.1, creatine phosphate 10, HEPES 5 and EGTA 1.1. No clamp was imposed and RPM should not vary by more than 10% otherwise cell was discarded. Firing was determined from a series of current injections (500 ms duration, 50 pA steps).

The Acb of rpS6^P−/− mice and wild-type littermates were homogenized in Krebs-Ringers bicarbonate buffer (125 mM NaCl, 1.4 mM KCl, 20 mM HEPES [pH 7.4], 5 mM NaHCO₃, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 1 mM CaCl₂) containing 1% BSA. Protein concentrations were determined using the BCA assay (Pierce). To measure ATP concentration, 100 μg of Acb lysates were used for the CellTiter-Glo Luminiscent Cell Viability kit (Promega) in a 96-well format following manufacturer’s instructions.

Complex I (EC 1.6.5.3) was measured by recording the decrease in absorbance due to oxidation of NADH (Roche) at 340 nm (ε = 6.2 mM⁻¹ cm⁻¹) in a POLARstar Omega plate reader (BMG Labtech) at 30°C in a volume of 1 ml. The Acb of rpS6^P−/− and wild-type littermates was dissected and stored at −80°C until further use. Tissue was homogenized with a glass potter in 150 μL of ice-cold Mir05 buffer (Mitochondrial Physiology Network: 0.5 mM EGTA, 3 mM MgCl₂, 60 mM lactobionate, 20 mM taurine, 10 mM KH₂PO₄, 20 mM HEPES, 110 mM sucrose and 1 g L−1 BSA). The homogenate was centrifuged at 500× g for 10 min at 4°C to remove debris. The supernatant was kept on ice for the assays. Tissue homogenate (200–250 μg protein) was added to the assay buffer (35 mM K₂HPO₄ pH = 7.2, 5 mM MgCl₂, 2.5 g L−1 BSA, 0.1 mM NADH) and the reaction was started by addition of 0.1 mM Decylubiquinone with changes in 340 nm absorbance (slope) measured for 3 min. Rotenone (12.5 μM) was added and monitoring carried for further 3 min. Complex I activity was taken as the rotenone-sensitive activity measured by the percent inhibition after addition of rotenone.

Citrate synthase (CS; E.C. 2.3.3.1) was measured by recording the increase in absorbance at 412 nm of the reaction of acetyl-CoA with Oxaloacetate (OAA) and the release of free CoA-SH to the colorimetric reagent 5,5-dithiobis(2-nitrobenzoate; DTNB; ε = 13.6 mM⁻¹ cm⁻¹) in a POLARstar Omega plate reader (BMG Labtech) at 37°C in a volume of 1 mL. Tissue homogenate (50–70 μg protein) was added to the assay buffer (20 mM Tris-HCl pH = 8, 0.1 mM DTNB, 0.1% Triton X-100) and the reaction was started by addition of 0.3 mM Acetyl-CoA, and 0.5 mM OAA with changes in 412 nm absorbance (slope) measured for 3 min. Assay conditions were controlled by using standard commercial CS (Sigma C3260) diluted 1:500 in PBS, pH = 7.0. Assay measurements were performed in duplicate. Brain samples were prepared as described above for Complex I.

Dendritic spine analysis was performed as previously described (Guegan et al., 2013). Briefly, rpS6^P−/− knockin mice and wild-type littermates were deeply anesthetized with pentobarbital (500 mg/kg, i.p.; Sanofi-Aventis) prior to rapid intracardiac perfusion, delivered with a peristaltic pump at 20 ml/min, with 10 ml of Na₂HPO₄/NaH₂PO₄/NaCl buffer (PBS) 0.1 M, pH 7.5, and followed by perfusion with 40 ml of 4% PFA in PBS 0.1 M, pH 7.5. Brains were postfixed in 4% PFA for 10 min and kept in PBS at 4°C. Brain coronal sections (100 μm) containing the Acb (Franklin and Paxinos, 2008; located between 1.54 mm and 0.98 mm anterior to Bregma) and DS (located between 1.42 mm and 0.14 mm anterior to Bregma) were obtained by using a vibratome (Leica) and stored at −20°C in a solution containing 30% (v/v) ethylene glycol, 30% (v/v) glycerol, and 0.1 M sodium phosphate buffer until they were processed for fluorescent labeling. Brain slices were labeled by ballistic delivery of fluorescent dye DiI (Molecular Probes, Eugene, OR, USA) using a gene gun apparatus (Helios Gene Gun System, Bio-Rad, Deutschland) and postfixed with PFA for 4 h at room temperature to further preserve structures and to allow the diffusion of the dye DiI. Sections were placed on microscope gelatine-coated slides and coverslipped with mounting medium. Acquisition and analysis of dendritic spine images was performed as described (Dumitriu et al., 2011, 2012). Z-Stacks were acquired using a 60 × 60 × 168 nm³ voxel size on an inverted Leica SP8 confocal laser scanning microscope at the Montpellier RIO Imaging Facility. Images were acquired with excitation at 561 nm and acquisition of the emission between 570 and 620 nm at a frequency of 400 Hz with a 63× 1.4 numerical aperture oil immersion objective. Images were averaged by four acquisitions to minimize noise. Laser intensity and photomultiplier gain were adjusted for each image to fill the entire range of the sensor and minimize the noise level. Secondary to tertiary dendrites of individual medium-sized spiny neurons were chosen for spine analysis Images were deconvolved with Huygens 3.5 (Scientific Volume Imaging) using an experimental Point Spread Function and spine analysis was performed using the semiautomated software NeuronStudio, which analyses dendritic length, dendritic width, spine number and spine head diameter in 3D. Parameters for dendritic spine quantification with NeuronStudio were: minimum neurite length = 10 μm; minimum spine height = 0.4 μm; maximum spine height = 3 μm; minimum stubby size = 20 voxels. Parameters for spine classification were: neck ratio = 1.1; thin ratio = 2.25; mushroom size = 0.4 μm. After NeuronStudio processing, verification of the dendritic spine densities quantification was performed in blind conditions and manually corrected if any errors in spine identification.

Anxiety-like behavior was measured as previously described (Busquets-Garcia et al., 2011).

Repetitive behaviors were assessed by marble burying activity. Mice were placed in a clean cage (46 × 20 × 14 cm) with 4 cm sawdust bedding overlayed by 20 glass marbles (15 mm diameter) equidistant in a 4 × 5 arrangement. Mice were allowed to explore the cage for 20 min and the number of marbles buried (>50% of the marble covered by the bedding) was counted.

Mice were suspended 50 cm above a cushioned pad using tape to attach their tails to a horizontal pole above the pad. Each mouse was tested for a 6-min trial. Latency to the first bout of immobility (defined as ≥ 5 s-long segment of time spent immobile) and the total time spent immobile during the trial were recorded. Immobility was defined as hanging passively without any movement of the head or paws.

Spontaneous alternation was measured in a Plexiglas Y-shaped maze with three identical arms (40 × 9 × 16 cm) at a 120° angle. Each individual mouse was placed in the center of the maze and allowed to freely explore for 5 min. A triad was defined as a set of three arm entries, when each entry was to a different arm of the maze. The number of arm entries and the number of triads were recorded. The percentage of alternation was calculated by dividing the number of triads by the number of possible alternations and then multiplying by 100.

A three-chamber arena was used to assess sociability, preference for social novelty, and social memory. On day 1, stranger target mice were habituated to the wire cups. On day 2, test mice (2-month-old males) were placed in the middle chamber and allowed to explore all the empty chambers of the apparatus freely for 10 min. Next, an unfamiliar mouse (Stranger#1, male C57BL/6 age matched) was introduced into one of the two side chambers, enclosed in a wire cage allowing only for the test mouse to initiate any social interaction. An identical empty wire cage was placed in the other side chamber. Following placement, the test mouse was allowed to explore the whole three-chamber arena for 10 min. At the end of the 10 min sociability test, a new unfamiliar mouse (Stranger#2, male C57BL/6 age matched) was placed in the previously unoccupied wire cage, and test mice were examined for an additional 10 min to assess preference for social novelty. The time spent in each compartment and the time spent sniffing the Stranger#1, Stranger#2, or empty wire cages were manually scored.

All statistical analyses were performed using one-way analysis of variance (ANOVA) for multiple comparisons, followed by Newman-Keuls post hoc test. Student t-test with equal variances was used for groups of two, when relevant. Statistical significance was determined as p < 0.05. Prism 5.0 software was used to perform statistical analyses. Locomotor activity was analyzed by matched two-way ANOVA (effect of time and effect of genotype), followed by Bonferroni’s multiple comparison test. All statistical analyses are available in Supplementary Table S1.

To determine the physiological role of rpS6 phosphorylation, we used rpS6 knockin mice (rpS6^P−/−), in which all phosphorylation sites were mutated to alanine residues (Supplementary Figure S1). We first monitored locomotor activity in non-stressful conditions (i.e., low luminosity) using circular corridors. A significant increase in horizontal locomotor activity was observed during the first 10 min in rpS6^P−/− mice (Figure 1A). In contrast, no differences were found in the number of rears (Figure 1B). This enhanced reactivity to a novel environment was further confirmed in mice video-tracked in an open field arena, as demonstrated by the significant increase in total distance traveled during the first 10 min in rpS6^P−/− mice (Figures 1C,D). This enhanced locomotor response was accompanied by an increased movement velocity (Figure 1E), a decrease in the total time spent immobile (Figure 1F) and the immobility frequency (Figure 1G). No difference in locomotor activity was found at longer time points (20 and 30 min) between genotypes in both the circular corridor and open field arena (Figure 1A and data not shown). The enhanced locomotion was not a result of novelty-induced anxiety since mice spent the same amount of time in the center of the open field (Figure 1H) and in the open arms in the elevated plus maze (Figures 1I,J). In the latter, the total arm entries were higher in rpS6^P−/− mice most likely due to the enhanced reactivity to novelty (Figure 1K). No differences between genotypes were found in other behavioral tasks including marble burying test, tail suspension test, Y-maze spontaneous alternation test and sociability as well as social novelty in the three-chamber test (Supplementary Figure S2). Together, our results indicate that novelty-induced locomotor activity is enhanced in rpS6^P−/− mice.

Altered size of several cell types has been reported in peripheral tissues of rpS6^P−/− mice (Ruvinsky et al., 2005, 2009). To determine whether rpS6 phosphorylation was also a critical determinant of neuronal size, we analyzed whether the brain was developed normally in adult rpS6 phosphorylation-deficient mice. No changes in the brain weight and size were observed between rpS6^P−/− mutant mice and wild-type littermates (Supplementary Figure S3). Histological analyses using the neuronal marker NeuN did not reveal any gross abnormality in cytoarchitecture in mutant mice (Supplementary Figure S3). No evident alterations of specific brain areas, including the DS and the Acb, were detected (Supplementary Figure S3). The expression levels of striatal proteins important for its functionality were also analyzed by immunofluorescence and western blots. No changes in dopamine D1 and D2 receptors expression were found (Figure 2A). The size, the distribution and the expression of medium-sized spiny neurons (DARPP-32) as well as different classes of striatal interneurons, including parvalbumin (PV), nNOS, calretinin (CR), and cholinergic (ChAT) interneurons were unaffected (Figures 2B,C and Supplementary Figure S4). Similarly, immunoblot analyses of striatal homogenates revealed no differences between genotypes in the expression levels of D2R, DARPP-32, dopamine transporter (DAT), guanine nucleotide-binding protein G(olf) subunit alpha (Gαolf), tyrosine hydroxylase (TH), monoamine oxidase A (MAO-A), vesicular monoamine transporter 2 (VMAT2) and norepinephrine transporter (NET) (Figures 2D,E).

To determine whether rpS6 phospho-deficiency could lead to discrete morphological changes, striatal slices of rpS6^P−/− and wild-type mice were labeled by ballistic delivery of fluorescent dye DiI. Analyses of dendritic spine density or types of spines (thin, stubby and mushroom) in labeled output neurons of the DS and Acb revealed no differences between genotypes (Figures 2F–H). In addition, equivalent contents of synaptophysin (Syp) and PSD95, pre- and post-synaptic markers respectively, were found (Figure 2I). Altogether, our results show that rpS6^P−/− mice display intact striatal morphology and normal levels of proteins involved in striatal function.

Enhanced rpS6 phosphorylation is frequently observed in various electrical and chemical models of synaptic plasticity (Biever et al., 2015b). To distinguish between D1- and D2-MSNs, we crossed rpS6^P−/− mice with Drd2-eGFP mice and we recorded both eGFP-positive (D2-MSNs) and eGFP-negative cells (most likely D1-MSNs) in the Acb and DS. Baseline electrophysiological parameters were similar in rpS6^P−/− mice compared to control, with the exception of accumbal D2-MSNs that showed a slightly elevated threshold for action potential (AP) generation. However, in the absence of altered RMP and input-output curves, this likely reflects a sampling error. Taken together, passive membrane properties, AP generation, and overall excitatory synaptic inputs were not affected by the genetic manipulation (Supplementary Figure S5). We next examined synaptic transmission at excitatory afferents onto MSNs after a challenge with a high-frequency stimulation (HFS) train. While the HFS protocol induced a LTP of the excitatory postsynaptic currents (EPSCs) in D2-MSNs in the Acb of wild-type mice, it failed to do so in rpS6^P−/− mice (Figure 3A). This effect was specific of the Acb since no differences between genotypes were observed after recording D2-MSNs in the DS (Figure 3B). Interestingly, similar results were obtained when the analysis was performed in D1-MSNs (Figures 3C,D). Together, our results indicate that rpS6 phosphorylation deficiency predominantly impacts synaptic plasticity in the Acb.

To investigate directly whether rpS6 phosphorylation participates in the fundamental control of mRNA translation in the nervous system, we performed polysome profile analysis on striatal lysates (comprising both the DS and the Acb) from rpS6^P−/− mice and wild-type littermates. As shown in Figure 4A, no difference in the polysome profiling was observed between genotypes. We also conducted polysome profiling analyses on frontal cortex or hippocampal lysates and failed to observe differences between genotypes (Supplementary Figure S6). Similar results were obtained when striatal de novo protein synthesis was assessed using an assay adapted from the ribopuromycylation method (David et al., 2012). Indeed, no difference in puromycin incorporation was found in striatal lysates of rpS6^P−/− mice compared wild-type littermates (Figure 4B). We next analyzed TOP-encoded proteins since increased rpS6 phosphorylation has been correlated with enhanced hippocampal TOP mRNA translation during LTP (Tsokas et al., 2005; Antion et al., 2008b). No changes in the TOP-encoded proteins rpS6 and eukaryotic translation elongation factor 1A (eEF1A) were observed in the striatum between genotypes (Figure 4C). Finally, no differences were found in the basal phosphorylation state of key components regulating the translational machinery including signaling pathways (mTORC1 and ERK), as well as translational initiation (eIF4E, eIF4G and eIF2α) and elongation (eEF2) factors (Figures 4D–F). Altogether, these results suggest that global striatal mRNA translation is not impaired in rpS6 phosphorylation deficient mice.

Despite the normal overall protein synthesis in rpS6^P−/− mice, we sought to assess whether basal rpS6 phosphorylation could regulate the translation of a specific subset of mRNAs, using the striatum as a test case. To address this issue, we performed polysome profiling of Acb and DS lysates followed by high-throughput RNAseq of heavy polysomal fractions, which contain the actively translated mRNAs (Figure 5A). Additionally, total mRNA abundance, reflecting the cytosolic steady-state mRNA levels, was also analyzed in whole Acb and DS lysates from both genotypes. We initially compared the fold changes of normalized read counts between genotypes in heavy polysomal fractions and whole lysates (containing cytosolic steady-state mRNAs). In the Acb, although wild-type and rpS6^P−/− mice displayed similar polysome profiles (Figure 5A), we identified 998 differentially expressed mRNAs, of which 497 were downregulated whereas 501 were upregulated in rpS6^P−/− mice. A heatmap depicting the top 100 differentially expressed genes between genotypes is shown in Figure 5B. Importantly, no changes in the normalized read counts of these 998 genes were observed in whole Acb lysates from rpS6^P−/− compared to wild-type littermates indicating that the described alterations did not result from changes in transcription or mRNA stability. Indeed, Ecscr was the only mRNA differentially modulated between the two genotypes. In contrast to the Acb, only 20 genes (Cwc22, Myl4, Mt2, Frrs1l, Ndufa3, Sncb, Usmg5, Atp5e, Bex2, Mt1, Ndufa1, Tomm7, Nap1l5, Pcp4, 2010107E04Rik, Pebp1, Tmsb10, Sat1, Selt, Timm8b) were differentially expressed in heavy polysomal fractions of DS lysates between genotypes, suggesting that the impact of phospho-rpS6 deficiency might be brain region-specific. To gain insight into the classes of genes differentially expressed in heavy polysomal fractions from Acb between genotypes, we performed GO analysis. Among the three domains covered by the ontology (biological process, cellular component and molecular function), terms related to mitochondrial functions are some of the most heightened, including “mitochondrial transport”, “electron transport chain”, “ATP synthesis coupled proton transport” (Figure 5C), “mitochondrial inner membrane”, “mitochondrial ribosome”, “mitochondrial respiratory chain” (Figure 5D), “electron carrier activity” and “NADH dehydrogenase (ubiquinone) activity” terms (Figure 5E), among others. Interestingly, all these terms are significantly de-enriched in rpS6^P−/− mice.

We next complemented our analysis with an additional method tool (Xtail pipeline, Xiao et al., 2016) allowing us to assess differential translation efficiency of each given mRNA on top of the transcriptional changes between genotypes. More precisely, a ratio of normalized gene counts from the RNAseq analysis on the “heavy polysomal fractions” and “whole lysates” was calculated for each gene. In line with our previous analysis, we identified 1484 genes with a statistically significant alteration in translational efficiency in the Acb (865 genes showing a decreased translational efficiency in rpS6^P−/− mice and 619 showing an increased translational efficiency; Figures 6A,B). In contrast, only 74 genes showed an altered heavy polysomes/whole lysate ratio between genotypes in the DS (39 genes showing a decreased translational efficiency in rpS6^P−/− mice and 35 showing an increased translational efficiency; Supplementary Figures S7A–C). We found a substantial overlap in the genes identified by our two analyses (Supplementary Figures S8A,B). In line with this observation, genes displaying differences in their translation efficiency belonged to similar GO terms when compared to those identified in our previous analysis (Figures 6C–E).

To further analyze the mitochondria-related genes dysregulation, we performed a hierarchical classification using the MitoCarta 2.0 inventory (Calvo et al., 2016). Indeed, among the 1071 mitochondria-related genes identified by RNAseq, 160 genes were differentially regulated in the heavy polysomal fraction between genotypes, of which 75% were downregulated and 25% upregulated in rpS6^P−/− mice (Figure 7A). A similar classification performed following Xtail analysis revealed 185 genes with altered translational efficiency, of which 62 and 123 have an enhanced and reduced translational efficiency, respectively (Figure 7B). Representative mitochondria-related genes found to be downregulated in Acb heavy polysomal fractions of rpS6^P−/− mice by RNAseq (Figure 7C) were validated by qRT-PCR analysis (Figure 7D). No changes at the transcriptional level of these genes were found in total mRNA lysates (Figure 7D) in agreement with RNAseq analysis.

Interestingly, we found that the majority of downregulated genes in knockin mice are part of the mitochondrial translational machinery (7 and 13 genes out of the 30 and 49 genes of the small and large mitochondrial ribosomal subunits, respectively; Figure 7A). On the other hand, this classification also revealed intriguing differences in genes belonging to the respiratory chain complex, since 49 genes (out of the 115 genes analyzed) were downregulated in rpS6^P−/− mice whereas only four were upregulated (Figure 7A). Although this bias was present in the five respiratory chain complexes, the effect was particularly evident for complex I (CI), in which 25 out of the 36 genes sequenced were downregulated in the knockin while only one was upregulated (Figure 7A).

Therefore, we next sought to analyze CI activity on total homogenates from the Acb of rpS6^P−/− mice and wild-type littermates. Despite the large number of downregulated genes encoding for CI proteins found in rpS6 phosphorylation-deficient mice, no alterations were observed in CI activity (Figure 7E), even after correction with the activity of CS (Figure 7F), a validated biomarker for mitochondrial density, as shown by the CI/CS ratio (Figure 7G). On the other hand, we also measured ATP levels since half of the complex V-related genes analyzed were downregulated in rpS6^P−/− mice. However, no differences in Acb ATP content were found between genotypes (Figure 7H). Together, our data indicate an altered translation, but not transcription, of a subset of mitochondria-related transcripts selectively in the Acb of rpS6^P−/− mice.