ORIGINAL RESEARCH published: 08 May 2020 doi: 10.3389/fnbeh.2020.00061 The Impact of the PI3K/Akt Signaling Pathway in Anxiety and Working Memory in Young and Middle-Aged PDK1 K465E Knock-In Mice Lydia Giménez-Llort 1,2 *, Mikel Santana-Santana 1,2 and José Ramón Bayascas 1,3 1 Institut de Neurociències, Universitat Autònoma de Barcelona, Barcelona, Spain, 2 Department of Psychiatry and Forensic Medicine, School of Medicine, Universitat Autònoma de Barcelona, Barcelona, Spain, 3 Department of Biochemistry and Molecular Biology, School of Medicine, Universitat Autònoma de Barcelona, Barcelona, Spain Dysfunction and dysregulation at the genetic, neural, and behavioral levels point at the fine-tuning of broadly spread networks as critical for a wide array of behaviors Edited by: Djoher Nora Abrous, and mental processes through the life span. This brain-based evidence, from basic to Université de Bordeaux, France behavioral neuroscience levels, is leading to a new conceptualization of mental health Reviewed by: and disease. Thus, the Research Domain Criteria considers phenotypic differences Wenqiang Chen, observed among disorders as explained by variations in the nature and degree of neural Joslin Diabetes Center, Harvard Medical School, United States circuitry disruptions, under the modulation of several developmental, compensatory, Pedro Bekinschtein, environmental, and epigenetic factors. In this context, we aimed to describe for the CONICET Institute of Cognitive and Translational Neuroscience (INCYT), first time the in vivo behavioral impact of tweaking the PI3K/Akt signaling pathway Argentina known to play an essential role in the regulation of cellular processes, leading to diverse *Correspondence: physiological responses. We explored the effects in young (YA, 3–4 months of age) Lydia Giménez-Llort and mature (MA, 11–14 months of age) male and female PDK1 K465E knock-in mice lidia.gimenez@uab.cat in a battery of tests under different anxiogenic conditions. The results evidenced that Specialty section: the double mutation of the PDK1 pleckstrin homology (PH) domain resulted in an This article was submitted to Learning and Memory, a section of enhancement of the negative valence system shown as an increase of responses of the journal Frontiers in Behavioral fear- and anxiety-like behaviors in anxiogenic situations. Interestingly, this seemed to Neuroscience be specific of YA and found regulated at middle age. In contrast, cognitive deficits, as Received: 20 January 2020 measured in a spatial working memory task, were found in both YA and MA mutants Accepted: 30 March 2020 and independently of the level of their anxious-like profiles. These distinct age- and Published: 08 May 2020 function-dependent impacts would be in agreement with the distinct cortical and limbic Citation: deficits in the Akt signaling in the brain we have recently described in these same Giménez-Llort L, Santana-Santana M and Bayascas JR (2020) The Impact animals. The elicitation of age- and neuronal-dependent specific patterns suggests of the PI3K/Akt Signaling Pathway in that fine-tuning the intensity of the PKB/Akt signal that enables diverse physiological Anxiety and Working Memory in Young and Middle-Aged response has also its in vivo translation into the negative valence system and age is a key PDK1 K465E Knock-In Mice. regulatory factor. Front. Behav. Neurosci. 14:61. doi: 10.3389/fnbeh.2020.00061 Keywords: RDoC, PI3K/Akt, signaling pathway, anxiety, cognition, animal model, aging, fine tuning Frontiers in Behavioral Neuroscience | www.frontiersin.org 1 May 2020 | Volume 14 | Article 61 Giménez-Llort et al. PI3K/Akt Signaling in Memory and Anxiety in Mice INTRODUCTION the phosphorylation of PKB/Akt isoforms, but leaving intact activation of the other AGC kinase family members (Bayascas The understanding of the age-dependent expression et al., 2008). These mice present a smaller body size and of psychiatric symptoms such as anxiety still demands insulin resistance, but their behavioral phenotype and its changes important efforts to unveil and scrutinize its biological and through age are still unknown (Bayascas et al., 2008). environmental basis through the life span. In the last decade, Therefore, the present study was aimed to explore the a new consideration of psychopathology is discussed in behavioral phenotype of the PDK1−/− PH domain knock-in terms of dysregulation and dysfunction in essential aspects mice. We recently reported that the deficits in the Akt signaling of behavior based on basic neuroscience and behavioral are pronounced both in the cortex and the hippocampus during science research (nimh.nih.gov/research/research-funded-by- young adulthood (3–4 months of age), but tend to be attenuated nimh/rdoc/index.shtml). The negative valence system (NVS; by middle age (11–14 months of age) in these mutant mice (Yang primarily responsible for responses to aversive situations or et al., 2018). In the present study, the behavioral and functional context, such as fear, anxiety, and loss) and cognitive system phenotype screening of these animals was analyzed. We used are two of the five tentative domains convened by the National a battery of four unconditional tests differing on the levels of Institute of Mental Health (2020) in its Research Domain fear and anxiety and where the sequence of behavioral events Criteria (RDoC) matrix. This matrix results from the crossword that are successively developed in an action program (Lát, 1973) between behavioral dimensions or functional constructs and involves cognitive function. Spontaneous exploratory behavior, seven different levels of analysis: from genes, molecules, cells, emotionality, bizarre and anxiety-like behaviors, habituation, as and neuronal circuits to physiology, behavior, and self-report well as working memory were evaluated to address the negative (Asher, 2010). In this context, while basic research on signal valence and cognitive systems involved. transduction is providing a refined close examination of the impact of cell membrane receptors and second messengers on MATERIALS AND METHODS cellular biochemistry and physiology, its downstream actions may be more difficult to characterize even more when related Analysis of the PDK1 K465E Mutation to age. For instance, the phosphatidylinositol 3-kinase (PI3K) Mutation of Lys465 in PDK1, which forms key interactions signaling pathway, which has been widely involved in controlling with the D3 and D4 phosphates of PtdIns(3, 4, 5)P3 , to a Glu neuronal development and function (Waite and Eickholt, 2010). residue abolished the binding of PDK1 to phosphoinositides and This pathway transmits the extracellular signals through the localization at the plasma membrane (Komander et al., 2004). 3-phosphoinositide-dependent protein kinase-1 (PDK1), an Prior to generating a knock-in mutation, the structure of the enzyme that emerged as the major transducer of PI3K actions. isolated PDK1(K465E) mutant PH domain was crystallized and PDK1 activates at least 23 AGC protein kinase family members analyzed to ensure that this mutation did not disrupt the overall besides Akt, the most popular effector of the pathway (Mora PH domain fold. The mutant protein was produced in bacteria et al., 2004; Pearce et al., 2010). Its functional role in cellular and expressed with yields similar to those for the wild-type biochemistry and physiology as well as cancer and metabolism PDK1 PH domain. The overall structure of the PDK1(K465E) has been largely explored; however, bottom-up approaches PH domain was unaffected by the mutation of Lys465, except to elucidate the impact of dysfunctional PI3K/Akt signaling for changes observed in the side-chain conformations of residues in behavior or how it differs through the aging process are located in the PtdIns(3,4,5)P3 -binding pocket. The K465E still scarce. mutation also significantly reduced the positively charged nature The behavioral readout resulting from modulation of the of the ligand-binding interface, which accounts for its inability to different sites of the PDK1 enzyme is currently studied using bind to phosphoinositides (Komander et al., 2004). new engineered animal models targeting key functional protein domains (Bayascas, 2008). Hypomorphic PDK1 mice, with a Generation of PDK1K465E/K465E Mice and reduced general activity of PDK1, showed several behavioral Genotyping Analysis differences related to anxiety and exploration in various tests The generation and the genotyping of the PDK1K465E/K465E (Leibrock et al., 2013). We previously generated neuronal- knock-in mice expressing the single-amino acid substitution specific conditional knock-in mice in which the expression of lysine 465 to glutamic acid in the PDK1 PH domain were of the PDK1 L155E mutant form was targeted to neuronal described previously (Bayascas et al., 2008). The mice were tissues by means of a Nestin-Cre-driven system (Tronche et al., subjected to PCR genotyping of the genomic DNA isolated from 1999). Disrupting the substrate docking site in PDK1 resulted ear biopsy using primers K465E F (50 -GGG TGA AGC ATG in the altered activation of several AGC kinases, but intact GAA TCT GTG TCT T) and K465E R (50 -GCC AGG ATA Akt activation (Cordón-Barris et al., 2016). These animals CCT AAG AGT ACC TAG AA). PCR amplification resulted in showed altered activation of several AGC kinases, but intact Akt a 196-bp product from the wild-type allele and a 236-bp product activation. Their phenotype was characterized by a smaller body from the targeted allele. size and showed sensorimotor problems, exacerbated disruptive behavior, and cognitive deficits. Oppositely, the PDK1-K465E Animals pleckstrin homology (PH) domain knock-in mice (hereinafter A total of 62 mice, PDK1−/− (n = 42) and PDK1+/+ [also referred PDK1−/− ) just have a mutation in the PH domain, only affecting to as wild type (WT), n = 20], including a pool of both sexes (50%) Frontiers in Behavioral Neuroscience | www.frontiersin.org 2 May 2020 | Volume 14 | Article 61 Giménez-Llort et al. PI3K/Akt Signaling in Memory and Anxiety in Mice and two maturation ages, YA (3- to 4-month-old young adults, graphical abstract, also including the conclusions, illustrates the n = 19) and MA (11- to 14-month-old mature adults, n = 43), methodological settings and procedures (Figure 1). were used. Mice were maintained at the Animal House Facility of the Day 0: Reflexes and Sensorimotor Tasks Universitat de Lleida under standard husbandry conditions Visual reflex and posterior leg extension reflex were measured (housed three to four per cage in 35 cm × 35 cm× 25-cm three times by holding the animals by the tail and slowly Macrolon cages, with food and water ad libitum, 22 ± 2◦ C, a 12-h lowering them toward a black surface. Motor coordination and light/dark cycle, and relative humidity of 50–60%). Behavioral equilibrium were assessed twice (20-s trials) in two consecutive assessments and data analysis were performed blind to the road tasks of increasing difficulty. The distance covered and the experiment, in a counterbalanced manner, in the light cycle, latency to fall off a wooden (1.3-cm width) and a metal wire from 09:00 to 13:00 h. All procedures were in accordance with (1-cm diameter) rods (both 1 m long) were recorded. the Spanish legislation on the ‘‘Protection of Animals Used for Experimental and Other Scientific Purposes’’ and the EU Day 1: Corner Test and Open Field Test Directive (2010/63/UE) on this subject. The study complies with Corner test (CT) was used to evaluate neophobia. The animals the ARRIVE guidelines developed by the NC3Rs and the aim to were individually placed in the center of a clean standard home reduce the number of animals used. cage filled with wood shave bedding and observed for 30 s. We measured the numbers of corners visited (CTc), the latency to Behavioral Assessments realize the first rearing (CTlatR), and the number of rearings Animals were behaviorally assessed for negative valence and (CTr). Once they completed the CT, the mice were placed in cognitive systems in a battery of tests composed of a corner test, the center of an illuminated (20 lx) open field (homemade an open field test, T-maze, and marble-burying test. Somatic woodwork, white box, 55 cm × 55 cm × 25 cm) and observed growth, as measured by body weight, as well as sensorimotor for 5 min. First, the ethogram of action programs (sequence of tasks were recorded on day 0 prior to the behavioral battery behavioral events) was analyzed. Thus, the duration of freezing of tests in order to monitor possible confounding factors. A behavior (latM, latency of movement) and the latency of the FIGURE 1 | Graphical abstract. Behavioral impact of the PI3K/Akt signaling pathway. Experimental design: 3-days battery of four behavioral tests to assess the Research Domain Criteria (RDoC), negative valence system (NV), and cognitive system (CS) in PDK1−/− and PDK1+/+ mice at young age (YA) and mature age (MA) are illustrated. Main findings are also indicated. Frontiers in Behavioral Neuroscience | www.frontiersin.org 3 May 2020 | Volume 14 | Article 61 Giménez-Llort et al. PI3K/Akt Signaling in Memory and Anxiety in Mice behavioral events that follow to it were recorded: leaving the symbols in the graphics. P-value < 0.05 was considered as central square (latC), reaching the periphery (thigmotaxis, latP), statistically significant. performing first wall rearing (latR), and first grooming (latG). Second, the time course and total levels of exploratory activity were measured as horizontal (C, number of crossings) and RESULTS vertical (Rw, rearing with wall support) locomotor activity. Somatic Growth/Body Weight Third, variables of emotionality included the number of The body weights of the animals (in grams)—YA PDK1−/− mice, defecations (Def), the presence of urine (Ur), and the grooming 18.67 ± 1.29; MA PDK1−/− mice, 22.90 ± 0.84; YA PDK1+/+ behavior, through its number (nG), latency (latG), and its total mice, 28.14 ± 1.92; YA PDK1+/+ mice, 32.00 ± 2.47—showed time (tG). Finally, as previously described (Baeta-Corral and genotype and age effects (G: F (1,58) = 30.692, P = 0.000; A: Giménez-Llort, 2014), we evaluated the presence of the following F (1,58) = 5.822, P = 0.019), with lower body weight of PDK1−/− bizarre behaviors: stereotyped rearings without wall support mice as compared to the age-matched WT and also for younger (Rc), recoils (Re), scratching (Sc), turning (T), stretch attendance animals compared to mature animals. Post hoc comparisons (Sa), jumping (Ju), and jerks (Jk). Bursting (grooming with a showed a higher body weight in WT mice as compared to pattern broken in its first step, where the animal is only washing PDK1−/− in both ages (P = 0.001 and P = 0.000 for YA hands) was measured through its number (nB), latency (latB), and MA, respectively). Only PDK1−/− MA exhibited a heavier and total time (tBG). body weight than their genotype-matched YA counterparts (P = 0.036). Day 2: T-Maze Spontaneous alternation of mice was tested in a T-shaped maze Reflexes and Sensorimotor Tasks (with arms 25 cm in length). The animals were placed inside No statistical differences of any type where found in the visual the ‘‘vertical’’ arm of the maze with the head facing the end task. All the mice obtained the maximum score possible. In wall. The performance was evaluated by determining with a the motor tasks, no genotype differences were found in any chronometer the time elapsed until the animal crossed (four- of them. Only, an age effect was observed in the second paw criteria) the intersection of thee three arms (latT) and the of two trials of the metal rod, with YA mice being able to time to complete the test (latF). The task finished when the mice maintain the equilibrium in the road during more time than visited the two arms of the maze. The entry of an already visited did the MA counterparts, as expected for their age-related arm in the trial before completing the test was considered an lower weight. error (eT). Corner Test Day 3: Marble-Burying Test Figures 2A,B illustrate the horizontal and vertical exploratory The procedure used was as previously described (Torres-Lista behaviors in the test for neophobia. The 2 × 2 GLM et al., 2015). The mice were placed individually facing the wall in a analysis showed genotype effects in the vertical activity, with standard home cage with six glass marbles (1 cm × 1 cm × 1 cm) increased latency of first rearing (G: F (1,58) = 8.350, P = 0.005) on a 5-cm-thick layer of clean woodcuttings. The marbles and consequent reduction in the number of rearings (G: where spaced in three rows of two marbles per row in one F (1,58) = 6.367, P = 0.014) in the PDK1−/− mice vs. the WT half of the cage. The mice were left in the cage with marbles (Figure 2B). Strong age effects also appeared, with higher for a 30-min period, after which the test was terminated by number of corners visited (A: F (1,58) = 20.719, P = 0.000) and removing the mice. The number of marbles buried, changed higher latency of first rearing (A: F (1,58) = 7.481, P = 0.008) in of position (partially buried or turned), and left intact (I) the MA vs. the YA group. Besides, a genotype × age interaction were measured. effect was found in the latency of rearing (G × A: F (1,58) = 4.072, P = 0.048) since the effect was mostly due to an increase in the Statistics PDK1−/− YA group. Statistical analyses were performed using SPSS 15.0 software. The post hoc comparisons analysis of the number of visited All data are presented as the mean ± SEM and illustrated corners indicated that the performances of both YA PDK1−/− as bars (pooled data by genotype or age) or as dots that (P = 0.000) and YA PDK1+/+ (P = 0.017) were lower than those illustrate the individual values in each group segregated by of their respective older age groups. Also, YA PDK1−/− mice sex and/or age, as indicated in the legends. To evaluate the showed lower number of rearings than their older MA PDK1−/− effects of genotype (G) and age (A) group, a 2 × 2 factorial group (P = 0.009) and as compared to the age-matched WT analysis design was applied. Differences were studied through mice (P = 0.009) due to an increased latency (P = 0.000 and multivariate general linear model analysis, followed by post P = 0.004, respectively). hoc Sidak test comparisons, when possible. For categorical variables, the Fisher’s exact test with 2 × 2 and 4 × 2 designs Open Field Test was used. Spearman’s correlation analyzed the body size and Figures 3, 4 depict the main behavioral domains, events, behavioral correlates. Graphics were made with GraphPad and units of analysis in the open field test, showing a Prism 6. To explore possible hints of sexual differences, males distinct performance on PDK1−/− compared to the WT (squares) and females (circles) were represented with different groups. Although no differences were found in the latency of Frontiers in Behavioral Neuroscience | www.frontiersin.org 4 May 2020 | Volume 14 | Article 61 Giménez-Llort et al. PI3K/Akt Signaling in Memory and Anxiety in Mice FIGURE 2 | Exploratory behaviors in the corner test in young adult (YA) and FIGURE 3 | Ethogram and emotionality in the open field test in young adult mature age (MA) PDK1−/− and age-matched wild-type (WT, PDK1+/+ ) mice. (YA) and mature age (MA) PDK1−/− and age-matched wild-type (WT; Data are expressed as mean ± SEM. Bars illustrate the age or the genotype PDK1+/+ ) mice. Data are expressed as the mean ± SEM or incidence. Bars groups, as indicated in the abscissae. Symbols are used to illustrate individual illustrate the genotype groups, as indicated in the Y -axis. Symbols are used values of females (white circles) and males (gray squares) of each genotype to illustrate the different groups or individual values, as depicted in the and age group (YA and MA), as indicated in the abscissae. Factorial analysis: legends or abscissae. Factorial analysis: G, genotype effect; A, age effect, G, genotype effect; A, age effect; G×A, genotype × age interaction effects, *P < 0.05, **P < 0.01. Post hoc test: genotype (g), g P < 0.05 vs. the *P < 0.05, **P < 0.01, ***P < 0.001. Post hoc test: genotype (g), corresponding WT (PDK1+/+ ) group; age (a), aa P < 0.01 vs. the gg P < 0.01 vs. the corresponding WT (PDK1+/+ ) group; age (a), a P < 0.05, corresponding YA of the same genotype. aa P < 0.01, aaa P < 0.001 vs. the corresponding YA of the same genotype. first movement or latency to leave the center (Figure 3A), stretch attendances (P = 0.014), reached the periphery later bizarre behaviors (Figure 4C), scarcely elicited in the WT, (P = 0.004), exhibited longer latency for wall rearing (P = 0.002), were conspicuously observed in the PDK1−/− mice. Thus, and a consequent low number of this behavior in the first an increased number of stretch attendance was found in the minute of the test (P = 0.013). As compared to the age-matched PDK1−/− genotype (G: F (1,58) = 5.460, P = 0.023; Figure 4C). WT animals, the stretch attendance of YA PDK1−/− was also A genotype main effect was found in the number of wall higher (P = 0.023) and the latency of wall rearing was delayed rearings in the second minute (G: F (1,58) = 6.126, P = 0.016; (P = 0.016), resulting in a lower number of rearing during the Figure 4B), with a decreased number for the PDK1−/− first (P = 0.028) and the second minute (P = 0.020) of the test mice. Genotype × age interaction effects were shown in the and, consequently, a lower total number of rearings (P = 0.031). latency (G × A: F (1,58) = 10.796, P = 0.002; Figure 3A) At mature ages, MA PDK1−/− exhibited faster apparition of and number of wall rearings in the first minute of the rearing (P = 0.034) as compared to their age-matched WT test (F (1,58) = 4.595, P = 0.036; Figure 4B), as well as counterparts. Finally, emotionality, as measured by the presence in the latency of grooming (F (1,58) = 4.139, P = 0.046; or not of urine and defecations, was analyzed by Fisher’s exact Figure 3A). test with 2 × 2 and 4 × 2 designs. Only for the urination The post hoc comparisons analysis showed that the ethogram were the two designs significant (P = 0.01458 and P = 0.032, and variables of analysis of YA PDK1−/− mice were indicative respectively). The detailed representation per group hints that of a worse performance as compared to their older MA the enhanced urination in PDK1−/− mice was mostly due to MA PDK1−/− counterparts. Thus, YA PDK1−/− exhibited more PDK1−/− mice. Frontiers in Behavioral Neuroscience | www.frontiersin.org 5 May 2020 | Volume 14 | Article 61 Giménez-Llort et al. PI3K/Akt Signaling in Memory and Anxiety in Mice FIGURE 5 | Spontaneous alternation in the T-maze in young adult (YA) and mature age (MA) PDK1−/− and age-matched wild-type (WT; PDK1+/+ ) mice. Data are expressed as the mean ± SEM. Symbols are used to illustrate individual values of females (white circles) and males (gray squares) for each genotype and age group. Factorial analysis: G, genotype effect, *P < 0.05. Post hoc test: age (a), a P < 0.05 vs. the corresponding YA of the same genotype. where MA PDK1−/− mice scarcely buried marbles as compared to their younger genetic counterparts (P = 0.002; Figure 6B). FIGURE 4 | Exploratory and bizarre behaviors in the open field test in young adult (YA) and mature age (MA) PDK1−/− and age-matched wild-type (WT; Body Weight and Behavioral Correlations PDK1+/+ ) mice. Data are expressed as the mean ± SEM. Bars illustrate the Spearman’s correlations between and within-test variables are genotype group, as indicated in the abscissae. Symbols are used to illustrate detailed in Supplementary Table S1 and summarized as follows. the different groups or individual values, as depicted in the legends or abscissae. Factorial analysis: G, genotype effect, *P < 0.05. Post hoc test: The body weight of the WT was correlated with the latency of genotype (g), g P < 0.05 vs. the corresponding WT (PDK1+/+ ) group; age (a), rearing in the CT (r = 0.558, P = 0.011), while in the PDK1−/− a P < 0.05 vs. the corresponding YA of the same genotype. genotype and the total sample of mice, weight was negatively correlated with the number of marbles buried (r = −0.312, P = 0.044 and r = −0.326, P = 0.01, respectively). A part of Spontaneous Alternation in the T-Maze the inter-test correlations between the different variables of a Although the latency to cross the intersection of thee three arms test, behavioral variables of the corner and the open field test and the latency to complete the task were similar among all were highly correlated and confirmed the results. Neophobia groups, a trend of reduced exploratory activity was shown by in the corner test strongly predicted the subsequent behavioral PDK1−/− mice (Figure 5A). A genotype effect was found in the ethogram (negative correlation with latencies: all r < −0.345, number of errors, with PDK1−/− mice exploring more visited P = 0.006), the vertical activity in the open field test (OF; arms (G: F (1,58) = 4.652, P = 0.035; Figure 5B) than did the WT positive correlations with wall rearing in the first 2 min of mice, where the incidence of errors was n = 1 for each age group. the test), and the performance in the marble test (r = −0.326, The post hoc comparisons analysis also showed an increased P = 0.01). Similarly, neophobia in the corner and open field number of errors in the YA PDK1−/− as compared to its older tests (ethogram) was correlated with the ethogram of behavioral genetic counterparts (P = 0.048; Figure 5B, right). events in the T-maze (all r > 0.253, P = 0.048). The number of errors in the T-maze was correlated with the time invested to Marble Burying Test explore the test (r = 0.528, P = 0.000) and the latency of rearing Analysis of the interaction with marbles (Figure 6) pointed in the corner test (r = 0.253, P = 0.048). Finally, we propose an at age effects, with a reduced number of marbles buried (A: illustration of meaningful correlations as wheels (see Figure 7), F (1,58) = 7.481, P = 0.009) and an increase in those left intact where distinct fine-tuning of PKI3 signaling in PDK1−/− and WT (A: F (1,58) = 4.967, P = 0.030) in the MA groups as compared to can be visually observed in an easier manner. YA mice (Figure 6A). The post hoc comparisons analysis revealed The data that support the findings of this study are available that this age pattern was more evident in the PDK1−/− genotype, from the corresponding author upon reasonable request. Frontiers in Behavioral Neuroscience | www.frontiersin.org 6 May 2020 | Volume 14 | Article 61 Giménez-Llort et al. PI3K/Akt Signaling in Memory and Anxiety in Mice FIGURE 6 | Marble burying in young adult (YA) and mature age (MA) PDK1−/− and age-matched wild-type (WT; PDK1+/+ ) mice. Data are expressed as the mean ± SEM. Bars are used to illustrate the different levels of marble burying, as depicted in the abscissae. +/+, PDK1+/+ genotype group; −/−, PDK1−/− genotype group. Factorial analysis: A, age effect, *P < 0.05, **P < 0.01. Post hoc test: age (a), aa P < 0.01, vs. the corresponding YA of the same genotype. FIGURE 7 | Wheels of fine-tuning for the PI3K/Akt signaling pathway. Illustration of the meaningful behavioral correlates among variables of the different tests in the whole sample of animals, PDK1−/− and age-matched wild-type (WT; PDK1+/+ ) mice. Spearman’s correlation analyzed the relationship between behavioral variables of the four behavioral tests and with somatic growth (body weight). *P value < 0.05 was taken as statistically significant. Positive (black) and negative (red) statistically significant correlations are plotted, with thickness of the dotted (*P < 0.05) and unbroken (**P < 0.01) lines being proportional to the statistically significant value of the correlation. Frontiers in Behavioral Neuroscience | www.frontiersin.org 7 May 2020 | Volume 14 | Article 61 Giménez-Llort et al. PI3K/Akt Signaling in Memory and Anxiety in Mice DISCUSSION other than Akt, respectively. Phenotypic characterization of two knock-in mouse strains expressing each of these mutations Brain-based evidence on the impact of dysregulation and allowed narrowing down the contribution of Akt compared to dysfunction, from basic neuroscience to behavioral levels, is the complementary PDK1 branch in mediating PI3K actions leading to a new conceptualization of mental health and disease. (Bayascas et al., 2008). The PDK1 K465E mice with reduced Thus, the strategic plan of NIMH is ‘‘to develop new ways of activations were viable and exhibited no adverse phenotypes classified disorders based on dimensions of observable behaviors other than being smaller (Bayascas et al., 2008; Zurashvili et al., and brain functions.’’ Far from due to discrete etiologies, the 2013). In contrast, the PDK1 L155E mice were embryonically RDoC Project considers phenotypic differences observed among lethal, and Cre-mediated bypass of the lethal period by targeting disorders as explained by variations in the nature and degree the expression of the mutant PDK1 L55E protein to the brain of neural circuitry disruptions, under the modulation of several led to neurodevelopmental disorders and disrupted behavior, developmental, compensatory, environmental, and epigenetic thereby highlighting the importance of the Akt-independent factors (Hyman, 2007; Morris and Cuthbert, 2012). Here, we actions of this signaling toolkit regarding brain development can now also add ‘‘and age/aging factors.’’ Thus, dysfunction and function (Bayascas et al., 2008). Nevertheless, in spite of and dysregulation at the genetic, neural, and behavioral the absence of overt phenotypes, the behavioral consequences of levels point at the fine-tuning of broadly spread networks as PDK1 K465E knock-in mutation and Akt activity ablation were critical for a wide array of behaviors and mental processes never explored. (Morris and Cuthbert, 2012). In this context, tweaking of the The present work describes, for the first time, the in vivo PI3K/PDK1/Akt signaling pathway in the PDK1 K65E knock-in effects of PDK1 mutation in the PH domain. We studied a set mice unveiled thresholds for its essential role in the regulation of male and female PDK1−/− PH-domain transgenic (PDK1−/− ) of cellular processes, leading to diverse physiological responses mice at two stages of adult maturation—young adulthood and (Zhou et al., 2014). middle age—and as compared to age-matched WT mice with PI3K plays fundamental roles in regulating virtually every normal aging. We have recently shown, at the biochemical physiological process related to cell growth, proliferation, level, that these animals exhibited pronounced deficits in Akt survival, and metabolism, where Akt was meant for decades signaling both in the cortex and the hippocampus during to represent the major, and perhaps unique, effector mediating young adulthood, but tend to be attenuated at middle age these cellular responses. However, since it was first identified (Yang et al., 2018). Here, behavioral and functional screening as the apical Akt kinase, PDK1 emerged as a major transducer for negative valence systems and cognitive systems indicated of PI3K actions by regulating a number of AGC kinase that the deregulation of this signaling pathway also has a family members besides Akt, including S6K, RSK, SGK, and higher impact at young adulthood, as shown by their increased PKC isoforms (Mora et al., 2004). The essentiality of this anxiety-like behavioral profile and working memory deficits, enzyme has been widely demonstrated by the severe phenotypes while changes in middle-aged animals were found restricted reported in different PDK1-deficient mice models (reviewed to emotionality. Somatic growth, as measured by body weight, in Bayascas, 2010). Thus, the PDK1/Akt pathway has been was reduced, as previously described (Bayascas et al., 2008; reported as intermediating in the role of neurotropic factors, Zurashvili et al., 2013). like brain-derived neurotrophic factor (BDNF), in the acquisition The corner test for mild neophobia to a familiar environment, of fear and mood disorders like depression and anxiety such as a standard home cage with new bedding, showed (Chen et al., 2006; Duman and Monteggia, 2006; Ou and delayed and reduced number of rearings in the mutant mice Gean, 2006; Martinowich et al., 2007). The reduced general (mostly in the YA animals), leading to genotype differences activity of PDK1 exhibited by the hypomorphic PDK1 mice and age interaction effects. The correlation analysis showed that (PDK1hm) results in several behavioral differences related to both horizontal and vertical activities in the corner test were anxiety and exploration assessed in various tests (Ackermann intercorrelated, mostly in the PDK1−/− genotype. In agreement, et al., 2008), while cognitively unimpaired Akt2 knockout a similar but smoother pattern was shown in the horizontal mice (Akt2−/− ) presented an anxiety- and depressive-like exploratory behavior of YA PDK1−/− , albeit it did not reach phenotype (Leibrock et al., 2013). More recently, the increase statistical significance. Differences in the horizontal activity due of Akt phosphorylation has been implicated in the rapid to the age factor were notorious, indicating lower neophobia antidepressant-like effects of different drugs in the basolateral response in middle-aged animals, independently of the genotype. amygdala, hippocampus, and the prefrontal cortex (Shi et al., This could be explained by long-term habituation to husbandry 2012; Wang et al., 2015; Tao et al., 2016). Conversely, mice with routines, reducing the chances of animals to perceive a new home high anxiety-related behaviors showed a stronger acquisition, cage as a potential threat. slower extinction, and spontaneous recovery of learned fear that The results of increased neophobia in the corner test were coincide with enhanced phosphorylation of Akt in the amygdala confirmed in a more anxiogenic test such as the open and (Yen et al., 2012). illuminated field. There, YA mutants showed a change in Biochemical and structural data permitted the rational design thigmotaxis, with a delay to reach the periphery and delayed of two new PDK1 single-amino acid mutations within either the latency for wall rearings. These changes in the behavioral PH domain, namely PDK1 K465E, or the PIF-pocket domain, sequence of events of the ethogram were due to the conspicuous termed L155E, impeding the activation of Akt or substrates appearance of stereotyped stretch attendance and stereotyped Frontiers in Behavioral Neuroscience | www.frontiersin.org 8 May 2020 | Volume 14 | Article 61 Giménez-Llort et al. PI3K/Akt Signaling in Memory and Anxiety in Mice rearing in mutant mice, independently of age. These risk (Miller and Cohen, 2001). Here, again, the worse performance assessment behaviors in the open field were scarcely exhibited was mostly observable in the YA mutant mice, whose brains by 6-month-old wild-type C57BL/6 × 129Sv mice, but were exhibited more pronounced deficits in Akt signaling in both found enhanced in animal models with anxious-like profiles the cortex and the hippocampus (Yang et al., 2018), key (Komander et al., 2004; Baeta-Corral and Giménez-Llort, 2014). neuroanatomical areas for these behaviors. These cognitive Their presence in those mutants, considered as bizarre behaviors deficits are interesting to note since the mutations related triggered by the extereoceptive anxiogenic stimuli of the open to the PDK1 and Akt signaling pathway, such as those in and illuminated arena, was related to high levels of anxiety Akt2 knockout mice, presented a higher anxiety-like and and was reversed by early neonatal handling (Baeta-Corral depressive-like behavior, but were cognitively intact (Ackermann and Giménez-Llort, 2014). In the case of PDK1fl/fl CRE+ et al., 2008). As mentioned above, middle-aged female PDK1fl/fl mice, middle-aged females also exhibited stereotyped stretching CRE+ mice also presented smaller size as well as sensorimotor as part of a more severe disruptive behavior that included problems, exacerbated bizarre behavior, and short-term memory vocalizations and hyperactivity and refusal to be handled and deficits in the Morris water maze (Cordón-Barris et al., tested (Cordón-Barris et al., 2016). Enhanced risk assessment or 2016). Therefore, while the anxious-like patterns seem to be vigilance is part of the pattern of responses to potential harm more sensitive to be affected, a tiny modulation and Akt (anxiety), included in the behavioral dimensions or constructs signal thresholds may determine distinct levels of cognitive within the NVS domain together with responses to acute threat system dysfunction. (fear) and to sustained threat, frustrative non-reward, and Similarly to the elevated T-maze (Asth et al., 2012), the loss (nimh.nih.gov). PDK1−/− mutation enhanced the anxious T-maze can be used as an animal model to simultaneously responses elicited in a potentially harmful environment. Among investigate memory and anxiety in mice. Thus, the latency to the variables of emotionality, the latency of grooming was reach the intersection of the arms is considered a measure of normal, but urination was present in the PDK1−/− genotype due copying with stress strategy, and it is used to measure anxiety to incidence in the middle-aged mutants. in very mild conditions (Guayerbas et al., 2000). In this respect, A striking parallelism in the ethogram was found in correlations with the variables related to neophobia in the corner middle-aged mutants and young adult WT, both in the temporal and open field tests were found when the whole sample was sequence of behavioral events and the level of their expression. analyzed and in each genotype. However, no differences were The action programs described by Lát (1973), which are found between genotypes, but a trend of shorter latency to reach consecutively developed by animals when confronting a new the intersection was shown by mutants, albeit it did not reach environment, were not distinguishable of those found at young statistical significance. adulthood, except for the first minute/s of the test when the Finally, mice were assessed in the marble burying test, which first action, which is related to fear, was elicited. Again, the has been successfully used for the pharmacological assessment freezing behavior of YA mutant mice evidenced an increased of obsessive–compulsive behaviors, anxiety, and psychosis drugs neophobic response, here expressed in terms of a 50% reduction (Jimenez-Gomez et al., 2011; de Brouwer et al., 2019). The of rearings during the first 2 min of the test. Freezing behavior pattern, with predominance in the incidence of ‘‘changed or and reduced total rearings were also shown by middle-aged partially buried’’ marbles and equal low numbers of marbles female PDK1fl/fl CRE+ mice, although an overall drop of activity buried and left intact, shown by all YA animals, resembled did not allow detecting differences in thigmotaxis (Cordón- that already reported by our laboratory in middle-aged males Barris et al., 2016). of the standard hybrid C57BL/6 × 129sv strain (Torres-Lista In the spontaneous alternation task in the black T-maze et al., 2015). Here, independently of the genotype, the MA resembling natural burrows, an innate alternation form of a animals showed a shift to the left, with increased numbers of win–shift behavior of foraging in the wild was elicited. In most marbles left intact in detriment of those buried as compared of the WT mice, alternation behavior was performed without to the burying pattern shown by YA animals. This age errors or only once in a couple of cases. In contrast, at both ages, effect was more clearly expressed in the PDK1−/− genotype, most mutant mice explored already visited areas of the maze, with the reduction of burying in MA vs. YA mice reaching which were considered errors attributed to working memory statistical significance. This drop in behavior resembled the problems and prefrontal cortex dysfunction, characteristic of effect obtained in wild-type mice after a chronic low 1-mg/kg, schizophrenia-like patterns (Goldman-Rakic, 1994). Alternation s.c, non-cataleptic dose of risperidone (Bardin et al., 2006; behavior has been shown to reflect short-term habituation Baeta-Corral and Giménez-Llort, 2014). A similar trend was of responding to stimuli based on their relative familiarity shown in middle-aged female PDK1fl/fl CRE+ mice, whose because of recent exposure (Sanderson and Bannerman, 2012). number of buried marbles was in detriment in favor of a Importantly, similarly to the rewarded alternation and win–shift statistically significant increase in the number of marbles only behavior on the radial arm maze, spontaneous alternation is changed of position or half buried as compared to their sensitive to hippocampal lesions (Deacon et al., 2002). The role age-matched wild-type counterparts (Cordón-Barris et al., 2016). of the prefrontal cortex is under discussion and considered by With respect to the integration of the results of the marble these authors (Sanderson and Bannerman, 2012) as being rather test with the other tests, meaningful correlations were found, involved in other more complex and demanding goal-directed with the number of buried marbles being directly related to behaviors where active maintenance of information is required neophobia in the corner test in both WT (negative correlation Frontiers in Behavioral Neuroscience | www.frontiersin.org 9 May 2020 | Volume 14 | Article 61 Giménez-Llort et al. PI3K/Akt Signaling in Memory and Anxiety in Mice with the number of rearings in the corner test) and the ETHICS STATEMENT PDK−/− (negative correlation with the number of visited corners) genotypes. This was in agreement with our previous The animal study was reviewed and approved by CEEAH. results in middle-aged standard hybrid C57BL/6 × 129sv (Baeta-Corral and Giménez-Llort, 2014), where the number of AUTHOR CONTRIBUTIONS buried marbles correlated with the latency of rearing in the corner test and the incidence of urine, the variable that was found LG-L designed and performed the behavioral tests and illustrated increased in MA PDK−/− mice. the graphical abstract and the proposed wheels of correlation. In summary, the double mutation of the PDK1 PH domain MS-S constructed and analyzed the matrix of data and illustrated resulted in an enhancement of NVS, shown as an increase of the results. JB provided the animals and financial support. All responses of fear- and anxiety-like behaviors in anxiogenic authors participated in the scientific discussions and contributed situations, which seemed to be specific of young adulthood to the writing of the manuscript. and found regulated at middle age, where only an increased emotionality was noted. In contrast, cognitive deficits, as measured in a spatial working memory task, were found in FUNDING both YA and MA PDK−/− mice and independently of the This work was funded by the Ministerio de Economía y level of their anxious-like profiles. This would be in agreement Competitividad (MINECO), Programa Estatal de Investigación, with the distinct cortical and hippocampal deficits in the Akt Desarrollo e Innovación Orientada a los Retos de la Sociedad signaling in the brain of these animals (Bayascas et al., 2008). (SAF2014-52813-R) to JB. The present results contribute to the in vivo characterization of the behavioral impact of Akt signaling pathway dysfunction and support the conceptualization of qualitative and quantitative ACKNOWLEDGMENTS variations of neural circuit disruptions underpinning phenotypic differences that are relevant to several mental disorders. We thank Jessica Pairada and Núria Moix (Estabulari de The elicitation of age-dependent specific patterns suggests Rosegadors of the Universitat de Lleida) for animal care. that fine-tuning the intensity of the Akt signal enables not only diverse physiological responses, as we have previously SUPPLEMENTARY MATERIAL demonstrated in these animals, but also their readout in vivo. Finally, the modulation by sex factor will deserve The Supplementary Material for this article can be found online further exploration. at: https://www.frontiersin.org/articles/10.3389/fnbeh.2020. 00061/full#supplementary-material. DATA AVAILABILITY STATEMENT TABLE S1 | Behavioral correlates among variables of the different test in the wholes sample of animals, PDK1−/− and age-matched WT (PDK1+/+ ) mice. The raw data supporting the conclusions of this article will be Spearman’s correlation analyzed the relationship between behavioral variables of made available by the authors, without undue reservation, to any the four behavioral tests and with somatic growth (body weight). qualified researcher. *P-value < 0.05 was taken as statistically significant; **P-value < 0.01. REFERENCES Bayascas, J. R. (2008). Dissecting the role of the 3-phosphoinositide-dependent protein kinase-1 (PDK1) signalling pathways. Cell Cycle 7, 2978–2982. Ackermann, T. F., Hörtnagl, H., Wolfer, D. P., Colacicco, G., Sohr, R., Lang, F., doi: 10.4161/cc.7.19.6810 et al. (2008). Phosphatidylinositide dependent kinase deficiency increases Bayascas, J. R. (2010). PDK1: the major transducer of PI 3-kinase actions. Curr. anxiety and decreases GABA and serotonin abundance in the amygdala. Cell. Top. Microbiol. Immunol. 346, 9–29. doi: 10.1007/82_2010_43 Physiol. Biochem. 22, 735–744. doi: 10.1159/000185557 Bayascas, J. R., Wullschleger, S., Sakamoto, K., García-Martínez, J. M., Clacher, C., Asher, J. (2010). Genes and Circuitry, not Just Clinical Observation, to Guide Komander, D., et al. (2008). Mutation of the PDK1 PH domain inhibits protein Classification for Research (Internet) NIMH. Available online at: https://www. kinase B/Akt, leading to small size and insulin resistance. Mol. Cell. Biol. 28, nimh.nih.gov/news/science-news/2010/genes-and-circuitry-not-just-clinical- 3258–3272. doi: 10.1128/mcb.02032-07 observation-to-guide-classification-for-research.shtml. Accessed 29 February, Chen, Z. Y., Deqiang, J., Bath, K. G., Ieraci, A., Khan, T., Siao, C. J., et al. 2020. (2006). Genetic variant BDNF (Val66Met) polymorphism alters anxiety- Asth, L., Lobão-Soares, B., André, E., Soares Vde, P., and Gavioli, E. C. (2012). related behavior. Science 314, 140–143. doi: 10.1126/science.1129663 The elevated T-maze task as an animal model to simultaneously investigate the Cordón-Barris, L., Pascual-Guiral, S., Yang, S., Giménez-Llort, L., Lope- effects of drugs on long-term memory and anxiety in mice. Brain Res. Bull. 87, Piedrafita, S., Niemeyer, C., et al. (2016). Mutation of the 3- phosphoinositide- 526–533. doi: 10.1016/j.brainresbull.2012.02.008 dependent protein kinase-1 (PDK1) substrate-docking site in the developing Baeta-Corral, R., and Giménez-Llort, L. (2014). Bizarre behaviors and risk brain causes microcephaly with abnormal brain morphogenesis independently assessment in 3xTg-AD mice at early stages of the disease. Behav. Brain Res. of Akt, leading to impaired cognition and disruptive behaviors. Mol. Cell. Biol. 258, 97–105. doi: 10.1016/j.bbr.2013.10.017 36, 2967–2982. doi: 10.1128/mcb.00230-16 Bardin, L., Kleven, M. S., Barret-Grévoz, C., Depoortère, R., and Newman- de Brouwer, G., Fick, A., Harvey, B. H., and Wolmarans, W. (2019). A critical Tancredi, A. (2006). Antipsychotic-like vs. cataleptogenic actions in mice of inquiry into marble-burying as a preclinical screening paradigm of relevance novel antipsychotics having D2 antagonist and 5-HT1A agonist properties. for anxiety and obsessive-compulsive disorder: mapping the way forward. Neuropsychopharmacology 31, 1869–1879. doi: 10.1038/sj.npp.1300940 Cogn. Affect. Behav. Neurosci. 19, 1–39. doi: 10.3758/s13415-018-00653-4 Frontiers in Behavioral Neuroscience | www.frontiersin.org 10 May 2020 | Volume 14 | Article 61 Giménez-Llort et al. PI3K/Akt Signaling in Memory and Anxiety in Mice Deacon, R. M., Bannerman, D. M., Kirby, B. P., Croucher, A., and Rawlins, J. N. Shi, H. S., Zhu, W. L., Liu, J. F., Luo, Y. X., Si, J. J., Wang, S. J., et al. (2012). (2002). Effects of cytotoxic hippocampal lesions in mice on a cognitive test PI3K/Akt signaling pathway in the basolateral amygdala mediates the rapid battery. Behav. Brain Res. 133, 57–68. doi: 10.1016/s0166-4328(01)00451-x antidepressant-like effects of trefoil factor 3. Neuropsychopharmacology 37, Duman, R. S., and Monteggia, L. M. (2006). A neurotrophic model for stress- 2671–2683. doi: 10.1038/npp.2012.131 related mood disorders. Biol. Psychiatry 59, 1116–1127. doi: 10.1016/j.biopsych. Tao, W., Dong, Y., Su, Q., Wang, H., Chen, Y., Xue, W., et al. (2016). 2006.02.013 Liquiritigenin reverses depression-like behavior in unpredictable chronic Goldman-Rakic, P. S. (1994). Working memory dysfunction in schizophrenia. mild stress-induced mice by regulating PI3K/Akt/mTOR mediated J. Neuropsychiatry Clin. Neurosci. 6, 348–357. doi: 10.1176/jnp.6.4.348 BDNF/TrkB pathway. Behav. Brain Res. 308, 177–186. doi: 10.1016/j.bbr.2016. Guayerbas, N., Sánchez, A. I., Gamallo, A., and De la Fuente, M. (2000). ‘‘Mouse 04.039 performance in an exploratory activity test as a longevity biomarker,’’ in Animal Torres-Lista, V., López-Pousa, S., and Giménez-Llort, L. (2015). Marble-burying is Research Welfare: A Partnership, eds J. A. TurMari and J. M. Orellana-Muriana enhanced in 3xTg-AD mice, can be reversed by risperidone and it is modulable (London, UK: Laboratory Animals), 159–161. by handling. Behav. Processes 116, 69–74. doi: 10.1016/j.beproc.2015. Hyman, S. E. (2007). Can neuroscience be integrated into the DSM-V? Nat. Rev. 05.001 Neurosci. 8, 725–732. doi: 10.1038/nrn2218 Tronche, F., Kellendonk, C., Kretz, O., Gass, P., Anlag, K., Orban, P. C., et al. Jimenez-Gomez, C., Osentoski, A., and Woods, J. H. (2011). Pharmacological (1999). Disruption of the glucocorticoid receptor gene in the nervous system evaluation of the adequacy of marble burying as an animal model of results in reduced anxiety. Nat. Genet. 23, 99–103. doi: 10.1038/12703 compulsion and/or anxiety. Behav. Pharmacol. 22, 711–713. doi: 10.1097/fbp. Waite, K., and Eickholt, J. (2010). The neurodevelopmental implications of PI3K 0b013e32834afebe signaling. Curr. Top. Microbiol. Immunol. 346, 245–265. doi: 10.1007/82_ Komander, D., Fairservice, A., Deak, M., Kular, G. S., Prescott, A. R., 2010_82 Peter Downes, C., et al. (2004). Structural insights into the regulation of Wang, W., Lu, Y., Xue, Z., Li, C., Wang, C., Zhao, X., et al. (2015). PDK1 by phosphoinositides and inositol phosphates. EMBO J. 23, 3918–3928. Rapid-acting antidepressant-like effects of acetyl-l-carnitine mediated by doi: 10.1038/sj.emboj.7600379 PI3K/AKT/BDNF/VGF signaling pathway in mice. Neuroscience 285, 281–291. Lát, J. (1973). The analysis of habituation. Acta Neurobiol. Exp. 33, 771–789. doi: 10.1016/j.neuroscience.2014.11.025 Leibrock, C., Ackermann, T. F., Hierlmeier, M., Lang, F., Borgwardt, S., and Yang, S., Pascual-Guiral, S., Ponce, R., Giménez-Llort, L., Baltrons, M. A., Lang, U. E. (2013). Akt2 deficiency is associated with anxiety and depressive Arancio, O., et al. (2018). Reducing the levels of Akt activation by PDK1 behavior in mice. Cell. Physiol. Biochem. 32, 766–777. doi: 10.1159/000354478 knock-in mutation protects neuronal cultures against synthetic amyloid-β Martinowich, K., Manji, H., and Lu, B. (2007). New insights into BDNF function peptides. Front. Aging Neurosci. 9:435. doi: 10.3389/fnagi.2017.00435 in depression and anxiety. Nat. Neurosci. 10, 1089–1093. doi: 10.1038/nn1971 Yen, Y. C., Mauch, C. P., Dahlhoff, M., Micale, V., Bunck, M., Sartori, S. B., et al. Miller, E. K., and Cohen, J. D. (2001). An integrative theory of prefrontal cortex (2012). Increased levels of conditioned fear and avoidance behavior coincide function. Annu. Rev. Neurosci. 24, 167–202. doi: 10.1146/annurev.neuro.24. with changes in phosphorylation of the protein kinase B (AKT) within the 1.167 amygdala in a mouse model of extremes in trait anxiety. Neurobiol. Learn. Mora, A., Komander, D., van Aalten, D. M., and Alessi, D. R. (2004). PDK1, the Mem. 98, 56–65. doi: 10.1016/j.nlm.2012.04.009 master regulator of AGC kinase signal transduction. Semin. Cell Dev. Biol. 15, Zhou, X., Cordon-Barris, L., Zurashvili, T., and Bayascas, J. R. (2014). Fine-tuning 161–170. doi: 10.1016/j.semcdb.2003.12.022 the intensity of the PKB/Akt signal enables diverse physiological responses. Cell Morris, S., and Cuthbert, R. N. (2012). Research Domain Criteria: cognitive Cycle 13, 3164–3168. doi: 10.4161/15384101.2014.962954 systems, neural circuits, and dimensions of behaviour. Dialogues Clin. Zurashvili, T., Cordón-Barris, L., Ruiz-Babot, G., Zhou, X., Lizcano, J. M., Neurosci. 14, 29–37. Available online at: https://www.ncbi.nlm.nih.gov/ Gómez, N., et al. (2013). Interaction of PDK1 with phosphoinositides is pubmed/22577302 essential for neuronal differentiation but dispensable for neuronal survival. National Institute of Mental Health. (2020). Research Domain Criteria. (Internet) Mol. Cell. Biol. 33, 1027–1040. doi: 10.1128/MCB.01052-12 NIHM. Available online at: https://www.nimh.nih.gov/research/research- funded-by-nimh/rdoc/index.shtml. Accessed 29 February, 2020. Conflict of Interest: The authors declare that the research was conducted in the Ou, L. C., and Gean, P. W. (2006). Regulation of amygdala-dependent learning absence of any commercial or financial relationships that could be construed as a by brain-derived neurotrophic factor is mediated by extracellular signal- potential conflict of interest. regulated kinase and phosphatidylinositol-3-kinase. Neuropsychopharmacology 31, 287–296. doi: 10.1038/sj.npp.1300830 Copyright © 2020 Giménez-Llort, Santana-Santana and Bayascas. This is an Pearce, L. R., Komander, D., and Alessi, D. R. (2010). The nuts and bolts of AGC open-access article distributed under the terms of the Creative Commons Attribution protein kinases. Nat. Rev. Mol. Cell Biol. 11, 9–22. doi: 10.1038/nrm2822 License (CC BY). The use, distribution or reproduction in other forums is permitted, Sanderson, D. J., and Bannerman, D. M. (2012). The role of habituation provided the original author(s) and the copyright owner(s) are credited and that the in hippocampus-dependent spatial working memory tasks: evidence from original publication in this journal is cited, in accordance with accepted academic GluA1 AMPA receptor subunit knockout mice. Hippocampus 22, 981–994. practice. No use, distribution or reproduction is permitted which does not comply doi: 10.1002/hipo.20896 with these terms. Frontiers in Behavioral Neuroscience | www.frontiersin.org 11 May 2020 | Volume 14 | Article 61