The mammalian target of rapamycin (mTOR) complex 2 (mTORC2) is a multimeric signaling unit that phosphorylates protein kinase B/Akt following hormonal and growth factor stimulation. Defective Akt phosphorylation at the mTORC2-catalyzed Ser473 site has been linked to schizophrenia. While human imaging and animal studies implicate a fundamental role for Akt signaling in prefrontal dopaminergic networks, the molecular mechanisms linking Akt phosphorylation to specific schizophrenia-related neurotransmission abnormalities have not yet been described. Importantly, current understanding of schizophrenia suggests that cortical decreases in DA neurotransmission and content, defined here as cortical hypodopaminergia, contribute to both the cognitive deficits and the negative symptoms characteristic of this disorder. We sought to identify a mechanism linking aberrant Akt signaling to these hallmarks of schizophrenia. We used conditional gene targeting in mice to eliminate the mTORC2 regulatory protein rictor in neurons, leading to impairments in neuronal Akt Ser473 phosphorylation. Rictor-null (KO) mice exhibit prepulse inhibition (PPI) deficits, a schizophrenia-associated behavior. In addition, they show reduced prefrontal dopamine (DA) content, elevated cortical norepinephrine (NE), unaltered cortical serotonin (5-HT), and enhanced expression of the NE transporter (NET). In the cortex, NET takes up both extracellular NE and DA. Thus, we propose that amplified NET function in rictor KO mice enhances accumulation of both NE and DA within the noradrenergic neuron. This phenomenon leads to conversion of DA to NE and ultimately supports both increased NE tissue content as well as a decrease in DA. In support of this hypothesis, NET blockade in rictor KO mice reversed cortical deficits in DA content and PPI, suggesting that dysregulation of DA homeostasis is driven by alteration in NET expression, which we show is ultimately influenced by Akt phosphorylation status. These data illuminate a molecular link, Akt regulation of NET, between the recognized association of Akt signaling deficits in schizophrenia with a specific mechanism for cortical hypodopaminergia and hypofunction. Additionally, our findings identify Akt as a novel modulator of monoamine homeostasis in the cortex.

Schizophrenia is a disorder caused by multiple genetic and environmental variables. Despite the disease's heterogeneous causes, current hypotheses suggest that dysfunction of dopamine signaling in the brain is one of the final common pathways involved. One gene that may be involved encodes the protein kinase Akt, which is regulated by hormones, growth factors, and neurotransmitter receptors. In this study, we examined the potential molecular mechanisms linking Akt dysregulation to cortical hypodopaminergia, and ultimately to the pathology of schizophrenia. Using transgenic technology, we generated a mouse model with defective neuronal Akt signaling. Neurochemical and behavioral phenotypes associated with schizophrenia include decreases in prefrontal dopamine signaling and deficits in sensorimotor gating, two phenotypes we observed in our transgenic animals. Further, we observed that impaired cortical Akt activity significantly enhanced norepinephrine transporter function. Interestingly, we found that by blocking this transporter, we could reverse the cortical hypodopaminergia and behavioral deficits seen in our transgenic mice. The norepinephrine transporter is a presynaptic membrane protein that is critical for maintaining both norepinephrine and cortical dopamine homeostasis. Taken together, this work supports the potential for targeting both Akt and the norepinephrine transporter for treating dopamine-related mood disorders.

Funding: This work is supported by National Institutes of Health grants DA14684 (to AG and LCD), MH058921 (to AG), DK085712 (to KDN and AG), MH084755 (to SDR), DK069927 (to KDN), and partially by DC009488 (to DBP), DK064857 (to KDN), resources of the Tennessee Valley Healthcare System, and by the Diabetes Research and Training Center (DRTC; DK20593, to AG and KDN). This work is partially supported by resources of the Vanderbilt University Silvio O. Conte Center for Neuroscience Research (MH078028, to PJG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

To test this hypothesis, we have generated an animal model in which mTORC2/Akt signaling down-regulation is achieved by neuronal deletion of a key mTORC2 regulatory subunit, rictor. We used a Cre-lox strategy to restrict the genetic deletion to neurons and bypass embryonic lethality associated with whole body deletion [21] . The goal of the present study is to test how alteration in Akt signaling affects DA homeostasis in the prefrontal cortex.

Termination of DA signaling at prefrontal synapses involves two mechanisms: degradation via enzymes including COMT, and clearance via the norepinephrine (NE) transporter (NET) [16] , [17] , [18] , which takes up both major brain catecholamines, DA and NE [17] , [19] . Interestingly, insulin administration, which stimulates mTORC2/Akt signaling, decreases NET transcription in brain, while hypoinsulinemia and decreased mTORC2/Akt signaling increases NET transcription [20] . Therefore, we hypothesized that dysregulation of mTORC2/Akt signaling may provide a mechanistic link to cortical hypodopaminergia. Specifically, we propose that reduced Akt activity mediates increased NET expression and increased DA clearance by noradrenergic neurons in cortex, a novel molecular mechanism that explains how Akt dysfunction contributes to a reduction in prefrontal DA.

While human imaging and animal studies implicate a fundamental role for Akt signaling in prefrontal DA networks, the molecular mechanisms linking mTORC2/Akt to schizophrenia-related neurotransmission abnormalities have been elusive [3] , [6] . Importantly, models of schizophrenia suggest that cortical deficits in DA neurotransmission and content, defined here as cortical hypodopaminergia, contribute to both the cognitive deficits and the negative symptoms characteristic of this disorder [14] . Consistent with this hypothesis, imaging studies reveal that genetic variation associated with low activity Akt alleles interact epistatically with catechol-O-methyltransferase (COMT), a gene responsible for degradation of prefrontal synaptic DA. Together, these interactions ultimately affect the fidelity of prefrontal networks in humans [3] by decreasing DA availability at prefrontal synapses [15] . Thus, a compelling hypothesis in schizophrenia is that impaired mTORC2/Akt signaling triggers aberrant regulation of DA homeostasis.

Putative evidence for a role of defects in mTORC2 signaling in mental illnesses preceded the discovery of the mTORC2 complex itself. Indeed, lithium, used to treat bipolar disorder, stimulates phosphorylation of Akt at Ser473, the mTORC2 phosphorylation site [7] . The link between mTORC2 signaling deficits and mental illness has been strengthened by seminal work demonstrating that certain antidepressants [8] , along with both typical [4] and atypical antipsychotics [9] , increase Akt Ser473 phosphorylation. Furthermore, findings of diminished Ser473 phosphorylation and/or activity in post-mortem brains of patients with schizophrenia [10] and depression [11] potentially fortify the association between dysregulation of mTORC2-Akt signaling and development of psychiatric illnesses, although these findings may be confounded by perimortem artifacts [12] . Recent observations that blunted Ser473 phosphorylation occurs in lymphocytes derived from patients with schizophrenia and psychosis-prone normal individuals [13] also support the plausibility that mTORC2-Akt deficits are involved in schizophrenia.

Mammalian target of rapamycin (mTOR) complex 2 (mTORC2) is one of two highly conserved protein kinases that are critical regulators of cell growth and metabolism. mTOR complex 1 (mTORC1) and mTORC2 are functionally distinct multiprotein complexes that are defined by their subunit composition, rapamycin sensitivity, and substrate selectivity. Raptor, mLST8, PRAS40, and mTOR comprise the rapamycin sensitive mTORC1 while the rapamycin insensitive mTORC2 contains rictor, mSIN1, mLST8, and mTOR. Two key substrates of mTORC1 are S6K and 4E-BP, which are important regulators of translation, while protein kinase B, also known as Akt, is the primary substrate of mTORC2 [1] . Specifically, mTORC2 is the kinase responsible for phosphorylation of Akt at serine residue 473, one of two key phosphorylation sites [1] . Akt is an extensively studied kinase that has been implicated in numerous disorders such as diabetes, obesity, cancer, and mental disorders such as schizophrenia [2] . Post-mortem, imaging, and genetic association studies in humans [3] , [4] , [5] reveal that Akt deficiencies are associated with schizophrenia. Genetic studies in rodents further corroborate the relationship between dysregulation in Akt signaling and disruptions in dopamine (DA)-associated behaviors linked to schizophrenia [4] , [6] .

Results

Rictor Deletion Attenuates Akt Ser473 Phosphorylation Akt deficiency is mechanistically linked to prefrontal cortex abnormalities and schizophrenia-linked phenotypes in several mouse models [6], although a clear molecular mechanism for how Akt regulates cortical function remains elusive. Here, we investigate the dopaminergic consequences of abolishing Akt phosphorylation at Ser473 in neurons by utilizing the Cre/LoxP system to delete rictor specifically in neurons. Mice were engineered with a floxed rictor allele, as previously described [21], and crossed with neuron-specific nestin gene (NES mice) Cre driver line. Validating our approach, rictor knockout (KO) mice lack rictor mRNA expression and rictor protein expression in a gene-dosage dependent manner within the brain and cortex (Figure S1; p<0.01 and p<0.05, respectively, by one-way ANOVA followed by Dunnett's test). Importantly for the current hypothesis, neuronal rictor deletion abolishes Akt phosphorylation at Ser473 within the cortex of rictor KO mice (Figure 1A; ***p<0.001 by one-way ANOVA Dunnett's test) compared to FLOX (floxed allele(s) in the absence of Cre), NES (Cre allele in the absence of a FLOX allele), and heterozygous rictor neuronal KO mice (HET). Phosphorylation of Akt at Thr308 (Figure 1B) and total levels of Akt (Figure 1C) within the cortex are not different among the genotypes, allowing a direct evaluation of the effects of Ser473 phosphorylation. Similarly, rictor deletion also abolishes Akt phosphorylation at Ser473 in other brain regions, such as the substantia nigra (SN)/ventral tegmental area (VTA) of rictor KO mice, while Thr308 phosphorylation and total levels of Akt are unaltered (data were normalized to control (FLOX) and reported as mean±s.e.m., p values by Student's t test; pAkt Ser473 FLOX = 100±13%, KO = 6±1%, p<0.001; pAkt Thr308 FLOX = 100±13%, KO = 89±8%, p = 0.49; total Akt FLOX = 100±13%, KO = 101±10%, p = 0.96). Furthermore, total protein levels of mTOR in the cortex are not altered by neuron-specific rictor KO (data were normalized to control (FLOX) and reported as mean±s.e.m.; FLOX = 100±9%, NES = 130±12%, HET = 118±12%, KO = 116±12%; p = 0.32 by one-way ANOVA followed by Dunnett's test). PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Figure 1. Neuronal rictor deletion specifically abolishes Akt phosphorylation at Ser473 in the cortex. (A) Phosphorylation of Akt on residue Ser473 in the cortex. (B) Phosphorylation of Akt at Thr308 and (C) total Akt in the cortex are similar for all genotypes. Shown are mean±s.e.m of optical densities as a percentage of FLOX control mice. Genotypes shown include animals expressing only nestin-CRE (NES), mice expressing two copies of the “floxed” rictor allele (FLOX) only, heterozygous mice which express nestin-CRE and a single copy of the “floxed” rictor allele (HET), and knockout mice expressing both nestin-CRE and two copies of the “floxed” rictor allele (KO). Total cortical protein extract was loaded in each lane. Representative immunoblots are shown, as probed with antibodies to phosphorylated Akt at Ser473 (A) Thr308 (B), total Akt (C), and actin to serve as a loading control. Samples n = 9–16. ***p<0.001 one-way ANOVA. https://doi.org/10.1371/journal.pbio.1000393.g001

Neuronal Rictor KO Mice Display Sensorimotor Gating Deficits Prepulse inhibition (PPI) behavior has long been identified as a promising phenotype for translational studies of schizophrenia owing to the direct parallels in expression between rodent and human subjects. While PPI deficits are present in psychiatric disorders other than schizophrenia, they have a clear heritable component in schizophrenic families and these deficits can be attenuated by antipsychotic drug administration. Furthermore, PPI deficits are also linked to the hypofunction of corticostriatal forebrain circuits that is characteristic of schizophrenia [22]. The PPI behavioral assay measures the degree to which the startle response elicited by a loud “pulse” sound is attenuated when immediately preceded by a non-startling “prepulse” sound. As such, it assays the degree to which a brief sensory trace can rapidly modify a subsequent motoric response, thereby representing a straightforward approach towards quantifying sensorimotor dysregulation, which is generally regarded as an endophenotype of schizophrenia. Since evidence suggests a role for Akt in schizophrenia, and rictor KO mice demonstrate profound deficits in Akt phosphorylation, we tested whether rictor KO mice display impaired PPI relative to FLOX control mice. No differences in startle responses elicited by a 94 dB sound pressure level (SPL) noise burst were observed, suggesting that hearing and gross motor function were similar between groups (Figure 2A; Student's t test; p = 0.44). By contrast, analysis of PPI behavior revealed clear differences between genotypes; startle reflex amplitude was inhibited at all prepulse intensities in FLOX control mice, with the amount of PPI increasing monotonically from 40 to 75 dB SPL (Figure 2B). In rictor KO mice, prepulse sound levels between 40 and 55 dB did not appreciably attenuate the startle reflex. PPI was not observed in rictor KO mice until prepulse levels >55 dB, albeit at a weaker level in comparison to FLOX mice. These differences gave rise to a significant reduction of PPI across prepulse sound levels (Figure 2B; *p<0.05 by ANOVA). This PPI deficit can also be expressed as a significant decrease in average PPI across all sound levels in rictor KO mice (Figure 2C; Student's t test, *p<0.05). Thus neuronal rictor deletion, with loss of Akt Ser473 phosphorylation, impairs the forebrain circuits critically involved with sensorimotor integration. Given the strong evidence for PPI deficits in schizophrenic patients, we hypothesize that this mouse model has the potential to lend novel insight into the molecular mechanisms by which Akt deficits contribute to the schizophrenic phenotype. PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Figure 2. Neuronal rictor deletion results in sensorimotor gating deficits as assayed by PPI. (A) No difference in startle reflex elicited by a 94 decibel (dB) sound pressure level (SPL) noise was observed between rictor KO and littermate FLOX control mice. (B) The percentage of PPI was reduced significantly across the entire dB range in rictor KO mice. (C) The average PPI across the entire dB range reveals a significant deficit in PPI in rictor KO mice; n = 6–7 animals. *p<0.05 Student's t test. https://doi.org/10.1371/journal.pbio.1000393.g002

Rictor KO Mice Display Hypodopaminergia in Rostral Cortex For almost 50 years, schizophrenia research has centered on dopaminergic signaling as a crucial component of the etiology of the disease [23],[24],[25]. In particular, the original “DA hypothesis” heavily supported the notion of excessive DA neurotransmission and DA content, defined here as hyperdopaminergia, within the brain [26],[27]. Subsequent revision of the hypothesis, however, has transformed thinking from a global hyperdopaminergia to a regional hyperdopaminergia within the striatum and dopaminergic hypofunction within the cortex [14],[28],[29]. While this conceptualization is an oversimplification of a highly complex disorder, we utilized this hypothesis to hone in on molecular mechanisms that contribute to cortical hypodopaminergia. Furthermore, previous studies have linked pre-frontal DA deficits with PPI deficits in animal models, and perhaps the PPI deficits observed in the rictor KO mice could be partially explained by alterations in cortical DA content [30],[31]. Thus, we investigated steady state levels of DA, serotonin (5-HT), and NE in a region of mouse brain that is roughly analogous to the human prefrontal cortex and contains areas such as the intralimbic, prelimbic, and anterior cingulate cortex. Interestingly, HPLC with electrochemical detection of DA, NE, and 5-HT in the PFC of rictor KO mice revealed striking alterations in the DA and NE tissue content of these animals, while 5-HT levels remained unchanged (Figure 3). While both DA and NE levels are significantly different in rictor KO mice, they change in opposite directions; NE tissue content is significantly increased (Figure 3A; **p<0.01 by one-way ANOVA followed by Dunnett's test) while DA levels are significantly decreased (Figure 3B; Student's t test, *p<0.05). In addition, NES mice show similar PFC DA content levels (6.4±0.2 ng/mg protein) as FLOX mice. Thus, rictor KO mice display a key feature of the “dopamine hypothesis” of schizophrenia, namely hypodopaminergia in the rostral cortex, which may explain the sensorimotor gating deficits described earlier. Importantly, 5-HT levels are unaltered in the cortex (Figure 3C; Student's t test, p = 0.26) indicating that rictor deletion does not simply result in global monoaminergic alterations but rather specific changes in the dopaminergic and noradrenergic systems. In addition, extracellular levels of DA were determined in the PFC of rictor KO mice by microdialysis. Under basal conditions, extracellular DA is not significantly different in rictor KO mice compared to FLOX controls (data are reported as pg of DA/µL, mean±s.e.m.; FLOX = 0.54±0.15, KO = 0.76±0.18, n = 4–5; p = 0.35 by Student's t test). While basal extracellular levels of DA are unaltered in rictor KO mice, these animals do display significant deficits in DA tissue content, suggesting that maintenance of DA homeostasis is perturbed. PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Figure 3. Monoamine content in the rostral cortex is significantly altered in rictor KO mice. Tissue content of (A) NE, (B) DA, and (C) serotonin (5-HT) in rostral cortical homogenates. Results are presented as mean±s.e.m ng/mg of protein, n = 4–10. *p<0.05; **p<0.01 Student's t test. https://doi.org/10.1371/journal.pbio.1000393.g003 While prefrontal hypodopaminergia has been linked to PPI deficits, other studies clearly demonstrate a link between striatal hyperdopaminergia and PPI deficits [32],[33]. Thus, we sought to determine if rictor deletion increases tissue levels of DA in the striatum or in projecting DA neurons from the SN and VTA. DA levels in the SN/VTA are not altered in rictor KO mice (data are reported as ng of DA/mg protein, mean±s.e.m.; FLOX = 4.4±0.5, KO = 4.8±0.4, p = 0.60 by Student's t test). Importantly, similar to the cortex, DA tissue content in the striatum of rictor KO mice is significantly decreased (data are reported as ng of DA/mg protein, mean±s.e.m.; FLOX = 101.2±7.7, KO = 80.1±3.4, *p<0.05 by Student's t test). Thus, our data indicate that the PPI deficits observed in rictor KO mice are likely to arise from impairments in DA neurotransmission.

Rictor Deletion Increases NET Expression and Function It is intriguing that DA content is decreased while NE content is increased in rictor KO mice. Importantly, decreases in mTORC2/Akt signaling induced by hypoinsulinemia have been shown to increase NET transcription [20]. Moreover, early studies and unpublished data from our laboratory implicate deficits in Akt signaling with not only increases in NET transcription but also acute increases in NET cell surface expression (i.e. intact Akt signaling decreases NET availability at the plasma membrane) [20],[34],[35],[36]. Thus, we predict that altered DA homeostasis in rictor KO mice is due to changes in NET cell surface expression mediated by impaired Akt phosphorylation. Importantly, unlike other brain regions where DAT is the primary mechanism for removing DA from the synapse, in cortex DAT contributes relatively little and NET performs the majority of DA clearance. Indeed NET has a higher affinity for DA than NE itself, but DA can also be degraded in the synapse by COMT [16],[17]. Given the pivotal role of rictor in Akt regulation and the role of Akt signaling in determining NET availability, we hypothesize that rictor KO mice will display aberrant NET regulation that sustains the alterations in NE and DA levels seen in the rostral cortex. As hypothesized, total cortical NET protein is increased approximately 2-fold in rictor KO mice compared to all other genotypes (Figure 4A; ***p<0.0001 by one-way ANOVA followed by Dunnett's test). Furthermore, biotinylation assays reveal that cell surface levels of NET are also significantly increased (Figure 4B; Student's t test, ***p<0.0001). Tyrosine hydroxylase (TH), a cytosolic protein, was detected exclusively in the total protein fraction but not in the surface fraction, indicating that the biotinylated fraction represents exclusively cell surface proteins. Finally, the striking enhancement in surface NET detected in rictor KO mice results in a significant increase in NET function as assayed by cortical synaptosomal NE uptake (Figure 4C; Student's t test, *p<0.05). The nearly 2-fold increase in cortical synaptosomal NE uptake was also observed for DA (Figure 4C; Student's t test, *p<0.05) indicating that rictor deletion increases DA clearance by NET in noradrenergic neurons and as a consequence reduces cortical DA content. Furthermore, DA content is not decreased due to increased degradation since COMT levels were not different in cortex compared to FLOX control mice (data were normalized to control (FLOX) and reported as mean±s.e.m.; FLOX = 100±8%, NES = 78±11%, HET = 130±24%, KO = 80±11%; p = 0.10 by one-way ANOVA followed by Dunnett's test). Thus, the increase in NET expression and function within the cortex of rictor KO mice has the potential to mechanistically explain both the increased NE tissue content and decreased cortical DA tissue content described earlier (Figure 3). Interestingly, we did not find a significant difference in serotonin transporter expression within the PFC of the rictor KO mice, as measured by citalopram binding (unpublished data). PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Figure 4. Neuronal rictor deletion results in increased NET expression and function. (A) NET protein levels in the cortex. Mean±s.e.m optical densities are shown as a percentage of FLOX control mice. Representative immunoblots are shown, as probed with antibodies to NET, and actin (loading control); n = 10. (B) Levels of surface NET as measured from the biotinylated fraction of cortical slices. Mean±s.e.m optical density is shown as a percentage of FLOX control mice. Representative immunoblots are shown, as probed with antibodies to NET, Na+/K+ ATPase to serve as plasma membrane/loading control (n = 3–5), and TH which is absent in the biotinylated fractions since it is a cytosolic protein. (C) [3H]NE and [3H]DA uptake into cortical synaptosomes of FLOX and KO mice. Mean±s.e.m uptake is shown as a percentage of uptake in FLOX control mice; n = 12–18. *p<0.05; ***p<0.001 Student's t test. https://doi.org/10.1371/journal.pbio.1000393.g004 While our data indicate that global neuronal mTORC2 dysfunction enhances NET function and induces cortical hypodopaminergia, we sought to demonstrate more specifically that these alterations could arise from downregulation of cortical Akt activity. Thus, we utilized the isoform specific Akt1 inhibitor [37],[38],[39] in cortical slices to show that Akt inhibition is capable of directly determining NET surface availability (Figure S2). Surface levels of NET are significantly enhanced in biotinylated cortical slices treated with the Akt1 inhibitor (Figure S2A; *p<0.05 by Student's t test). Importantly, the levels of Akt Ser473 phosphorylation are substantially diminished in samples of these inhibitor treated slices (Figure S2B; ***p<0.0001). Together, these data support the notion that Akt stimulated regulation of the transporter occurs not only at the level of transcription, as is seen in rictor KO mice, but also at the level of transporter trafficking. Furthermore, the ability of Akt inhibition to enhance NET surface expression in cortical slices indicates that all the molecular machinery necessary for this rictor/Akt regulation of NET is intact within the PFC and thus is consistent with our hypothesis that altered cortical monoamine homeostasis via aberrant NET regulation underlies PPI deficits in rictor KO mice. We hypothesize that amplified NET function in rictor KO mice enhances the accumulation of both NE and DA within the noradrenergic neuron leading to conversion of DA to NE and ultimately supporting both increased NE tissue content and a state of hypodopaminergia. Such a mechanism within the prefrontal cortex provides an elegant molecular mechanism linking Akt hypophosphorylation to both cortical hypodopaminergia and PPI deficits, two key hallmarks of schizophrenia.

Midbrain Dopaminergic Neurons and Cortical Monoaminergic Projections Are Unaltered in Rictor KO Mice Considering the widespread function of Akt and its role in cell growth and proliferation, we next sought to demonstrate that the changes in DA and NE levels within the cortex were specifically due to increased NET expression rather than global changes in the number or projections of dopaminergic and noradrenergic neurons. While rictor KO mice do display a gross reduction in brain size, similar to what is seen for Akt3 deficient mice (brain weight normalized to body weight; FLOX 2.49±0.18% compared to KO 1.63±0.10%; p<0.0005 by one-way ANOVA followed by Dunnett's test), coronal brain sections stained for TH revealed no significant alterations in dopaminergic cell number within the VTA or SN (Figure 5A and 5C–5D; VTA p = 0.82 by one-way ANOVA, SN p = 0.53 by one-way ANOVA). Furthermore, TH staining of dopaminergic and noradrenergic projections within the cortex do not reveal any gross alterations among the groups (Figure 5B). The immunostaining was confirmed with Western blot analysis of total cortical TH protein levels (Figure 5E; p = 0.20 by one-way ANOVA). Other markers of dopaminergic neurons in the cortex were not significantly altered in the rictor KO mice such as total levels of D2 DA receptors (data were normalized to control (FLOX) and reported as mean±s.e.m.; FLOX = 100±5%, NES = 95±13%, HET = 96±9%, KO = 108±18%; p = 0.85 by one-way ANOVA followed by Dunnett's test). These data demonstrate that the DA and NE systems in the rictor KO mice are not globally altered. Therefore, we propose that the changes in cortical NE and DA tissue content can be primarily accounted for by the specific enhancement of NET expression in noradrenergic neurons. PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Figure 5. TH staining and expression in the midbrain and cortex is similar in NES, FLOX, and KO mice. TH immunoreactivity in the (A) midbrain substantia nigra (SN) and ventral tegmental area (VTA) and the (B) cortex. Scale bars = 50 µm. Coronal brain sections were stained with TH antibody and cell counts of TH+ cells were taken. Cell counts are similar in NES, FLOX, and KO matched mice in both the (C) VTA and the (D) SN. Mean±s.e.m TH+ cells/mm2 are shown; n = 6. (E) TH protein levels in the cortex. Mean±s.e.m optical densities are shown as a percentage of FLOX control mice; n = 19–22. One-way ANOVA analysis reveals no significant difference between genotypes. https://doi.org/10.1371/journal.pbio.1000393.g005