Saccharomyces cerevisiae Mep2. We show that Mep2 variants lacking the C-terminal tail conserve the ability to induce filamentation, revealing that signaling can proceed in the absence of exclusive binding of a putative partner to the largest cytosolic domain of the protein. Our data support that filamentation signaling requires the conformational changes accompanying substrate translocation through the pore crossing the hydrophobic core of Mep2. pHluorin reporter assays show that the transport activity of Mep2 and of non-signaling Mep1 differently affect yeast cytosolic pH in vivo, and that the unique pore variant Mep2H194E, with apparent uncoupling of transport and signaling functions, acquires increased ability of acidification. Functional characterization in Xenopus oocytes reveals that Mep2 mediates electroneutral substrate translocation while Mep1 performs electrogenic transport. Our findings highlight that the Mep2-dependent filamentation induction is connected to its specific transport mechanism, suggesting a role of pH in signal mediation. Finally, we show that the signaling process is conserved for the Mep2 protein from the human pathogen Candida albicans.The dimorphic transition from the yeast to the filamentous form of growth allows cells to explore their environment for more suitable niches and is often crucial for the virulence of pathogenic fungi. In contrast to their Mep1/3 paralogues, fungal Mep2-type ammonium transport proteins of the conserved Mep-Amt-Rh family have been assigned an additional receptor role required to trigger the filamentation signal in response to ammonium scarcity. Here, genetic, kinetic and structure-function analyses were used to shed light on the poorly characterized signaling role of  Saccharomyces cerevisiae Mep2 in the signal mediation leading to filamentation. If signaling partners exist, they should also bind to cytosolic loops and/or membrane-embedded domains. The capacity of Mep2 to enable filamentation is closely intertwined to the mechanism of substrate translocation through the pore of the hydrophobic core of the protein. In Xenopus oocytes, the transport activity of non-signaling Mep1 is electrogenic while it is electroneutral for Mep2, the latter likely translocating the weak base NH3, but not the proton released after NH4+ recognition and depronotation. We propose that given consequences of a Mep2-specific transport process, such as an intracellular pH modification, could be the underlying cause of the filamentation signal ensured by Mep2-type proteins.Fungal Mep2-type ammonium transport proteins of the conserved Mep-Amt-Rh family that includes human Rhesus factors are specifically required to allow filamentation in response to ammonium limitation. These proteins were therefore assigned a receptor role while the underlying mechanism of signal transduction remains poorly understood. The “transceptor” property has subsequently been proposed to concern transporters of all kind of micro- and macro- nutrients in eukaryotes, from fungi to human. However, bringing the firm demonstration of their existence remains challenging as variants with full uncoupling of transport and receptor functions are difficult to obtain. Our data question the involvement of the C-terminal extremity of  Candida albicans, diploid cells of baker yeast Saccharomyces cerevisiae respond to nitrogen limitation by switching to an unipolar mode of budding thereby producing pseudohyphae that invade the agar medium, likely to scavenge the environment for nutrients , 1915, 1mepΔ diploid cells, lacking all three MEP genes, but expressing Mep2 variants bearing domain deletions were assayed for filamentation efficiency by dropping high-density cell suspensions onto a specific SLAD medium containing a limiting ammonium concentration (100 μM) and following the appearance of protruding filaments. A similar SHAD medium containing a higher ammonium concentration (1 mM), conditions in which filamentation is repressed, was used to check for potential constitutive induction of filamentation. In parallel, the transport functionality of Mep2 variants was evaluated by two ways. We followed the growth development of single colonies from low-density suspensions of cells streaked on SLAD to test the capacity of cells to form colonies in the condition of filamentation induction and on SHAD. We also measured the ammonium removal ability of cells exponentially growing in a similar liquid medium, SHPD, containing a non-limiting concentration of proline (0.1%) as sole nitrogen source. Although ammonium removal in SHPD can’t be directly compared to growth tests on limiting SLAD, the assay gives an indication on the functionality of Mep2 variants in optimal conditions. The results of the distinct assays are recapitulated in CΔ428–431, Mep2CΔ428–449, Mep2CΔ434–449) was affected on SLAD and SHAD  of Mep2 had no major impact on the cells filamentation efficiency and on ammonium removal rate . The Mep2CΔ469–485 remained largely sensitive to the Npr1 absence, and unable to allow filamentation  lying along the conducting pore and playing a key role in transport in Mep-Amt-Rh proteins  from the extracellular medium compared to native Mep2, while the Mep2G349C variant conserving the CTD was even more efficient (G349C variant was visible on SHAD (1 mM ammonium) at day 7  using a defined growth condition previously shown to enable the recording of both Mep activities within minutes We are sorry that we cannot be more positive about your manuscript at this stage, and we hope that you find the reviews helpful and constructive. Please do not hesitate to contact us if you have any concerns or questions.Yours sincerely,Joeheitm001@duke.edu)Joseph Heitman, MD, PhD  and electrochemical current (in Xenopus). These support a difference in transport mechanism between the signaling Mep2 and non-signaling Mep1 proteins, concluding that Mep1 transports the ammonia molecule with its proton and Mep2 beings in only the proton.These are intriguing conclusions based on solid and rigorous experiments. They do not identify the elusive signaling mechanism, but the paper does lead to new directions for understanding it. The electrophysiology in Xenopus is particularly persuasive. The subject matter and approach are appropriate for PLoS Genetics. A few, mostly minor, comments are below.1. The weakest experimental aspect of the paper is the measurements of cytosolic pH. There are believable differences between Mep1 and Mep2, but they are quite small. This is likely because of the proton pumps in the plasma and vacuolar membranes. Perhaps if these were inhibited, pharmacologically or genetically, the differences would be enhanced and the confounding pH recovery phase reduced.2. The genetic experiments are thorough, but it is a bit confusing to keep track of all the mutants and what they are. A cartoon of the linear protein sequence, with the TMs, the enhancer, AI domains, and the position of the point mutants would be very helpful. Perhaps this could be added at the top of Fig. 1?3. Are they sure that the Mep2 enhancer domain mutants, which have reduced transport and signaling activity are stably expressed? A Western would be confirmatory .4. Some of the figures  have many constructs tested and it is challenging to really sort out which line represents which mutant . A consistent color scheme would help greatly. Alternatively, splitting this into two figures with the enhancer domain mutants in one and the others in another might help.Reviewer #2: Filamentation is the critical process for fungal development and pathogenicity of many fungal pathogens. In many fungi, filamentation is triggered by nitrogen starvation and therefore Mep2, ammonium permease, has been implicated in the process. A question remains whether Mep2 has a ‘transceptor’ activity for the filamentation process. That is to say, does Mep2 not only sense ammonium, but also transport it for filamentation? In this paper, Brito et al. tackled this question through genetic, molecular, and structure analyses of yeast Mep2. Through domain deletion experiments of Mep2 in triple mep deletion mutant background, the authors clearly demonstrated that ammonium transport function is tightly correlated with filamentation efficiency. They showed that the phosphomimetic mutation S457D in the C-terminal autoinhibitory domain of Mep2 did not lock Mep2 as a constitutive filamentation active form and it still required substrate translocation. Previously, it has been shown that the C-terminal domain (CTD) plays a role in tuning the transport activity of Mep2. Here the authors demonstrated that the CTD domain is not absolutely required for filamentation induction, because the H199Y and G349C suppressor mutation in the transmembrane domains can trigger filamentation even without the CTD domain. Based on the analysis based on reported X-ray structure of ScMep2, H199 and G349, which are evolutionarily divergent and conserved among other fungal orthologs, respectively, are facing each other. Subsequently, they observed the different impact of Mep1 and Mep2 on cytosolic pH. Supporting this, electrophysiology analyses using heterologous expression of Mep1 and Mep2 in Xenopus oocytes demonstrated that non-filamentation signaling Mep1 mediated electrogenic transport while Mep2 mediated electroneutral substrate translocation.Overall, this study provides very informative and valuable data for confirming the transceptor activity of Mep2 required for yeast filamentation. Here the authors clearly demonstrated that the Mep2 transceptor not only functions as ammonium transporter and a signaling receptor for filamentation, even without involvement of cytosolic partner proteins. So intracellular pH change can be a signal mediator for yeast filamentation. The manuscript is very well written and most of conclusions are nicely supported by a sophisticated set of experiments. I have the following minor comments.[Minor comments]Each paragraph should be indented throughout the manuscript.The first paragraph of the Introduction section is too long. It needs to be divided into two paragraphs (line 65?)Line 76-78: The meaning of this sentence is unclear. Please clarify it with more detailed explanation.Lines 132-141: A graphic illustration of each domain in the Mep2 protein  should make readers better understood. In addition, any previously known model describing how Mep2 protein works in association with Npr1 kinase could help readers too.It seems that only two independent experiments were performed for Figure 1B and Figure 3C experiments. For statistical analysis, at least three biological independent experiments should be performed and proper statistical analysis should be done.The graphs in Figure 2B are very difficult to see. Please use colored lines with more distinct symbols.Each Figure 5 graph should be separately plotted at 2 mM and 20 mM NH3, as the two data are overlapped and hard to discriminate.Reviewer #3: This study aimed at elucidating which specific aspects of the ammonium permease Mep2, as opposed to its paralogs Mep1 and Mep3, are responsible for its ability to induce filamentous growth in response to nitrogen limitation in the budding yeast Saccharomyces cerevisiae. By testing the ability of mutated forms of Mep2 to mediate ammonium transport and promote filamentation, the authors confirmed their previous observation that the induction of filamentous growth by Mep2 generally correlates with its ammonium transport activity. The C-terminal cytoplasmic tail of Mep2 was found to be dispensable for the induction of filamentous growth by some mutated forms of the protein, and the authors conclude that it does not represent a signaling domain. Electrophysiological studies showed that ammonium transport by Mep2 is electroneutral  whereas transport by Mep1 is electrogenic . Accordingly, the authors observed a slight increase in the cytosolic pH after ammonium addition to cells containing Mep2, which was weaker in cells producing only Mep1. The authors conclude that pH modification is the mechanism by which Mep2 induces filamentous growth.This is a very interesting study that addresses an unresolved question, how Mep2 and related ammonium permeases in other fungi induce morphogenesis.There are a number of issues that in my opinion affect the authors’ main conclusions and should be considered.Comments1) It is difficult to envisage that the very minor (less than 0.05 pH units) and presumably short-term (minutes) increase in the cytosolic pH after the addition of ammonium to the cells is the trigger for filamentous growth, considering that the induction of filamentous growth occurs after several days of growth under limiting nitrogen conditions; one would assume that the cells maintain intracellular pH homeostasis under these conditions. Furthermore, the experiments shown in Fig. 5 were performed in the presence of 2 mM ammonium, a concentration that suppressed filamentous growth in the present study . At the low concentrations of ammonium that induce filamentation, one would expect a still smaller, if any, effect on cytosolic pH by Mep2-mediated ammonium uptake. Of note, Mep2 also induces filamentous growth at low concentrations of other nitrogen sources, and this is believed to be due to Mep2-mediated retrieval of excreted ammonium. Under these conditions, even less extracellular ammonium will be present that can be taken up by Mep2 and affect the cytosolic pH. Although the authors also state that conformational changes in Mep2 that occur during ammonium transport are involved in signaling, the sentences at the end of the abstract, author summary, introduction, and discussion convey the message that Mep2-mediated pH changes are the key signal that induces filamentation.2) The presentation of the results shown in Figs. 1 to 3 could be considerably improved. For one thing, the growth differences between the various strains are not evident in Fig. 1C, Fig. 2B and E, and Fig. 3B, although the triple mepΔ mutants should not grow on SLAD medium. I assume that the triple mepΔ mutants and some of their derivatives formed tiny colonies, and the pictures were taken at different magnifications to show the colony borders, although this is nowhere stated in the manuscript. Growth differences are illustrated in Fig. 1A, Fig. 2A and D, and Fig. 3A, but these pictures appear not very reliable, as is evident from the fact that the mep2Δ single mutant seems to have a growth defect on SLAD medium that is rescued after reintroduction of wild-type MEP2 . I suggest the following modifications to these experiments and their presentation:a) Growth and filamentation of the various strains on solid media should be simultaneously illustrated by showing representative single colonies of all strains at equal magnification. I notice that the filamentation phenotype of the wild type on SLAD medium was generally weak in the authors’ experiments, with only occasional pseudohyphae at the colony borders, whereas in other studies, S. cerevisiae colonies grown on SLAD plates are completely surrounded by a filamentous fringe .b) The effect on filamentation by all Mep2 variants should also be tested in a mep2Δ single mutant background  to exclude an influence of reduced growth on filamentation.c) The doubling times of the strains in liquid SLAD medium should be determined to allow a better quantification of the effect of the different Mep2 mutations on growth for comparison with the effect on ammonium transport, which was also determined for cells growing exponentially in liquid medium.d) Finally, I found it very difficult to find the data for some strains in Fig. 1B, because the symbols cannot be easily distinguished. This could be amended by using colored symbols.3) I think that the authors should tone down their conclusion in the abstract that the C-terminal tail of Mep2 is not required for the induction of filamentation. Despite the fact that the additional H199Y mutation in the truncated Mep2S426stop protein restored ammonium uptake to wild-type levels , filamentation was extremely poor . Therefore, the function of the C-terminal tail is not only to upregulate substrate translocation, but it is also required for normal signaling at wild-type transport levels. Signaling may require interactions between the C-terminal tail and other cytoplasmic domains of Mep2 , and conformational changes caused by the H199Y mutation may have allowed some signaling to occur in the absence of these interactions .4) The H194E mutation is highly interesting in that it abolishes filamentation, although it increases ammonium transport, a striking exception to the generally observed correlation between ammonium transport and signaling capacity by Mep2. The authors argue that the H194E mutation altered the mechanism of ammonium transport by Mep2 into a Mep1-like mechanism, as already suggested in their previous study. Unfortunately, this mutated protein could not be successfully produced in oocytes, which would have allowed to test if the mutation changed ammonium transport from electroneutral to electrogenic . As this is a critical aspect of the present study, I think that the authors should at least test if Mep2 with the H194E mutation shows a Mep1-like behavior instead of a Mep2-like behavior with regard to cytosolic alkalinization . If this test can reliably distinguish between the two transport mechanisms, the authors could prove or refute this hypothesis.5) Although the study focuses on Mep2 of S. cerevisiae, the paper implies that the conclusions about Mep2 function derived from this study extend to its homologs in other fungi. Nevertheless, the authors point out striking differences between the Mep2 proteins of S. cerevisiae and C. albicans, which may hint to different signaling mechanisms by the two proteins. In C. albicans, the C-terminal cytoplasmic tail of Mep2 is required for the induction of filamentous growth even when ammonium transport by mutant proteins appears normal. Mep2 proteins that were C-terminally truncated after amino acid 440 and 423 allowed wild-type growth and exhibited comparable ammonium transport activity. However, while the former promoted hyperfilamentation , the latter was unable to induce filamentation. According to the model proposed in the present study, the removal of the 17 additional amino acids should have altered the mechanism of ammonium transport (to a Mep1-like mechanism) to explain the loss of signaling. The C. albicans mutants are available and could be tested and compared in the same way as the S. cerevisiae mutants. Since the authors repeatedly highlight the relevance of the transition from yeast to filamentous growth for the virulence of pathogenic fungi (and our understanding of its regulation), it seems worthwile to perform and include these experiments.Reviewer #4: In this manuscript the authors build on their previous studies of the Mep2 transceptor from Saccharomyces cerevisiae. Three main findings are presented: that signalling is linked to transport activity, that the Mep2 CTD is not required for signalling and that signalling involves pH changes within the cytosol. In support of the latter, the authors present evidence that the Mep2 transceptor and the non-signalling Mep1 transporter induce different cytosolic pH profiles. Furthermore, the authors establish that transport by Mep2 is electroneutral whereas transport by Mep1 is electrogenic. Together these data support the model that Mep2 imports ammonia gas that raises cytosolic pH. This is an important finding. What is disappointing is that the authors have not gone one step further and tested some predictions of this model. For example, if the Mep2 dependent pH change regulates signalling then Mep2 variants that transport but do not signal should generate a different pH profile. This may be because transport by the variant is electrogenic or if it remains electroneutral the amount of imported ammonia differs from that imported by wild-type Mep2. Too little or too much ammonia may result in a pH change that does not support signalling. The authors could test the effect on cytosolic pH of the Mep2H194E variant as they have determined that transport by it is electroneutral. Similarly, the hyperactive Mep2G349C variant would be expected to generate a different pH profile from that of the Mep2H194E variant. Other points of discussion could include those Mep2 homologues that act as signalling proteins in S. cerevisiae, such as Ump2 from U. maydis. These should have the same impact as Mep2 on cytosolic pH via electroneutral transport when expressed in S. cerevisiae.Minor commentslines 131-198The fact that certain Mep2 variants are more active in the absence of Npr1 may be due to their increased expression and could be tested. A previous study by the authors  established that the Mep2 levels are higher in the absence of Npr1.lines 191-131The authors conclude that the Mep2S457D variant does not lock Mep2 into a constitutively active conformation as it does not induce filamentation when cells are grown on SHAD plates. Expression of this variant would be expected to be reduced under SHAD conditions. Wild-type Mep2 needs to be highly expressed if it is to signal in response to low ammonium. A variant locked in a constitutively active conformation may therefore loose its ability to signal if expressed at a low level. The relative levels of Mep2S457D in response to SLAD and SHAD should be determined.**********Have all data underlying the figures and results presented in the manuscript been provided?PLOS Geneticsdata availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.Large-scale datasets should be made available via a public repository as described in the Reviewer #1: YesReviewer #2: YesReviewer #3: YesReviewer #4: Yes**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article  and your responses to reviewer comments. If eligible, we will contact you to opt in or out. *Dear Anna Maria,Thank you very much for submitting your revised Research Article entitled 'Yeast filamentation signaling is connected to a specific substrate translocation mechanism of the Mep2 transceptor' to PLOS Genetics. Your manuscript was fully evaluated at the editorial level and by the same four independent peer reviewers who reviewed the original submission. The reviewers appreciated the attention to an important topic but identified some aspects of the manuscript that should be improved.  See further comments from the associate editor below.We therefore ask you to modify the manuscript according to the review recommendations before we can consider your manuscript for acceptance. Your revisions should address the specific points made by each reviewer.In addition we ask that you:1) Provide a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.archive. 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Reviewer 3 raised a few remaining issues that we request you address by revision, and this constitutes now minor compared to major revision. We appreciate your efforts to respond to the reviews, and improve the manuscript.Reviewer's Responses to QuestionsComments to the Authors:Please note here if the review is uploaded as an attachment.Reviewer #1: I am impressed by the extensive and thoughtful changes to the manuscript in response to the previous suggestions, particularly the clever pHluorin fusion experiments. I have no further reservations regarding the manuscript, though the authors may want to re-edit the paragraph at the bottom of page 17, as there are several typos.Reviewer #2: The authors nicely addressed all of my comments in this revised manuscript. I do not have any more comments. Nice work!Reviewer #3: In their revised manuscript, the authors went to great lengths to address the reviewers’ comments. Therefore, I would not request additional experiments, but there are several issues that in my opinion should be clarified.1) I still have difficulties to see how the short-term changes in intracellular pH that were observed upon addition of ammonium to proline-grown cells can be responsible for the induction of filamentous growth after several days under nitrogen-limiting conditions, as the cells should re-establish and maintain intracellular pH homeostasis. The authors may also explain why there is an initial drop in intracellular pH upon ammonium uptake, the reason for which is not clear to me. Although Mep1 transports the proton together with ammonia, one would expect that ammonia is immediately protonated again within the cell, so that there is no net increase in the intracellular concentration of protons by Mep1-mediated ammonium transport. In the case of Mep2, ammonia transport without the dissociated proton should cause an initial intracellular alkalinisation, because cytosolic protons are captured by the internalized ammonia. Which mechanisms are responsible for the actually observed pH changes?2) The authors point out that some Mep2 CTD variants showed an even better complementation efficiency in the absence of Npr1 than in its presence, and that this correlated with an improved ammonium removal rate in SHPD and also with an increased capacity to induce filamentation on SLAD (Page 9). This appears to be the case for the variants shown in the upper panels of Fig. 1C. However, the Δ442-449, Δ442-485, and Δ450-485 variants, which all confer a strong hyperfilamentous phenotype in the absence of Npr1  do not show better ammonium uptake in the absence of Npr1 than in its presence . If anything, the uptake rate is slightly lower in the absence of Npr1 (this is now better visible in the revised figure). Therefore, stronger filamentation does not correlate with more efficient ammonium uptake in these cases.3) In a similar vein, the repeatedly emphasized correlation between transport and filamentation efficiencies does also not hold in several other cases. The H199Y S426stop mutation did not impair transport , but almost completely abolished filamentation . Furthermore, the G349C mutation strongly increased transport  without resulting in enhanced filamentation in NPR1 wild-type cells . This should be more critically discussed.4) In my previous comments I had suggested that the authors compare the mode of ammonium transport by two different C-terminally truncated C. albicans Mep2 proteins, one of which promotes strong hyperfilamentation, while the removal of few additional amino acids abolished filamentation without altering transport efficiency. According to the model proposed in the present study, the additional truncation should have altered the mode of transport from Mep2-like to Mep1-like. Instead, the authors compared wild-type CaMep2 and a mutated form, in which H1 of the conserved histidine-twin in the conducting pore was changed into glutamate found in Mep1-like proteins, upon heterologous expression in S. cerevisiae. The finding that the mutation abolished filamentation is taken as evidence that a conserved transport mechanism explains the ability of both ScMep2 ans CaMep2 to promote filamentous growth. However, the authors disregard (see line 451) that the mutated Mep2 restored growth of SCmepΔ triple mutants on SLAD plates much less efficiently than did wild-type CaMep2 . It is likely that the mutated protein was less stable than the wild-type protein, explaining its inability to promote filamentation.Reviewer #4: -Associate editor note, this reviewer did not have detailed comments for the authors and found the manuscript improved and suitable for acceptance.**********Have all data underlying the figures and results presented in the manuscript been provided?PLOS Geneticsdata availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.Large-scale datasets should be made available via a public repository as described in the Reviewer #1: YesReviewer #2: YesReviewer #3: YesReviewer #4: Yes**********what does this mean?). 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PLOS Genetics may also choose to issue a press release for your article. If there's anything the journal should know or you'd like more information, please get in touch via  12 Feb 2020PGENETICS-D-19-00855R2 Yeast filamentation signaling is connected to a specific substrate translocation mechanism of the Mep2 transceptor Dear Dr Marini, We are pleased to inform you that your manuscript entitled "Yeast filamentation signaling is connected to a specific substrate translocation mechanism of the Mep2 transceptor" has been formally accepted for publication in PLOS Genetics! Your manuscript is now with our production department and you will be notified of the publication date in due course.The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any errors. 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Akt isoform, conditional brain-specific Akt1, and double Akt1/3 mutant mice in behaviors relevant to neuropsychiatric disorders. Because sex is a determinant of these disorders but poorly understood, sex was an experimental variable in our design. Our studies revealed AKT isoform- and sex-specific effects on anxiety, spatial and contextual memory, and fear extinction. In Akt1 mutant males, viral-mediated AKT1 restoration in the prefrontal cortex rescued extinction phenotypes. We identified a novel role for AKT2 and overlapping roles for AKT1 and AKT3 in long-term memory. Finally, we found that sex-specific behavior effects were not mediated by AKT expression or activation differences between sexes. These results highlight sex as a biological variable and isoform- or cell type-specific AKT signaling as potential targets for improving treatment of neuropsychiatric disorders.AKT is implicated in neurological disorders. AKT has three isoforms, AKT1/AKT2/AKT3, with brain cell type-specific expression that may differentially influence behavior. Therefore, we examined single  Psychiatric disorders, including schizophrenia, major depressive disorder (MDD), and anxiety, are important public health burdens with large societal and economic costs . GeneticAkt1 haplotypes have been identified in schizophrenic patients (The protein kinase B (PKB/AKT) family of serine/threonine kinases is involved in numerous neuromolecular signaling processes  and has patients . Additiopatients . AKT is patients . Therefopatients . AdditioAkt1 mutants. Therefore, this study provides novel evidence demonstrating AKT isoform-specific regulation of neural function in a sex-specific fashion. Combined with the fact that AKT isoforms are expressed in a cell type-specific fashion, these findings improve understanding of how AKT activity is specifically involved in distinct behaviors and neurobiological processes relevant to diagnoses and therapies directed against AKT signaling pathways for treating psychiatric disorders.To address these gaps in our knowledge of AKT signaling related to psychiatric disorders, we selectively removed AKT isoforms from the mouse brain using complementary genetic approaches, generating mutant mice with single-isoform deficiencies in AKT1, AKT2, or AKT3; selective loss of AKT1 in forebrain excitatory neurons; or double loss of AKT1 and AKT3 isoforms. With these mice, we performed murine behavioral assays that are models for affective behavior, spatial learning and memory, and associative fear memory and extinction. The behavioral results provide multiple lines of evidence demonstrating specific and overlapping roles for AKT isoforms in the activity of neural circuits that may model brain processes impacted in neuropsychiatric disorders. On balance, AKT1 exerts the strongest individual isoform effects on cognition and behavior, influencing males selectively in many of the tests we performed. Like AKT1, AKT2 affects anxiety-like behavior in a sex-specific fashion and impacts contextual memory. AKT3 exerted no effects on its own but in the background of AKT1 deficiency, enhanced deficits we observed in Akt mutant mice with a basic locomotor assessment using the open field arena (OFA) test. Besides locomotor activity, OFA assesses anxiety-like behavior by measuring time spent exploring the periphery or exposed center of the arena (Akt1 KO mice spend significantly less time exploring the center compared with wild-type (WT) littermate controls  testing, which examines anxiety-like behavior and exploratory drive in rodents . Time spcontrols . EPM tescontrols , indicatale mice  while Akther sex  in the EAkt to spatial learning and memory using the Morris Water Maze (MWM)  . In thiscontrols , indicat in  KO mice . For Aktin males . In Akt3 effects .AKT mutations and single nucleotide polymorphisms (SNP)s have been linked to psychiatric disorders like schizophrenia  is restored in neurons . Akt1 KO+AKT1PFC males showed a significant difference in extinction learning from Akt1 KO-sham mice (p=0.047) but not from WT-sham mice (p=0.875), consistent with the idea that PFC AKT1 activity underlies the extinction effects we observed in Akt1 KO mice. We also found a significant difference in extinction LTM between Akt1 KO-sham and WT-sham groups (Akt1 KO+AKT1PFC groups (post-hoc comparison p=0.985), supporting the notion that PFC-expressed AKT1 rescued Akt1 KO effects on extinction LTM. Finally, we examined extinction renewal following PFC AKT1 expression. We did not observe a significant difference between WT-sham and Akt1 KO+AKT1PFC groups (Akt1 KO-sham groups (post-hoc comparison p=0.375), as we did with WT and Akt1 KO mice in the previous experiment  for normal display , we next KO mice . To do t control . We conf control . Further neurons , both ex neurons . We founKO males . HoweverKO males , Akt1 KOperiment . HoweverAkt1 deficiency led to the phenotypes we observed in Akt1 KO males, we generated conditional KO mice with Cre-mediated Akt1 removal (Akt1 cKO) from excitatory neurons of the forebrain, including the amygdala and PFC   . When we the OFA  or EPM =3.427, p=0.005; Akt2 KO t(12)=2.282, p=0.042; Akt3 KO t(8)=11.79; p<0.0001), with the greatest reduction in Akt3 KO mice. Interestingly, when we examined AKT activation with the pan-pAKT antibody, we found increased pAKT levels in male Akt1 KO mice compared with WT controls =3.507, p=0.004; Akt2 KO t(10)=2.253, p=0.048; Akt3 KO t(7)=11.77, p<0.0001). Similarly, we found increased pan-pAKT levels in female Akt1 KO mice compared with WT controls , but pAKT1 levels were significantly higher in both male and female Akt3 KO mice compared with WT controls , and schizophrenia . HippocaAkt deficiency in several murine behaviors and expands previous knowledge on the contribution of AKT isoforms. For affective behaviors, no prior study had examined Akt1 KO mice. An earlier study reported higher anxiogenic-like behaviors in Akt2 KO mice, as we report, but did not examine sex-specific effects  littermates. Apart from previously reported significant size differences in Akt1 and Akt3 mutant backgrounds  mice for escribed . To geneckground . Becausekgrounds  and lowe KO mice , KO and Timeline: A schematic representation of the experimental timeline is shown in 2) with 180 lux overhead lighting and 55 dB white noise present for their entire duration in the testing room. Data were collected and analyzed using the Ethovision XT video tracking system , with the center zone defined as the area 10 cm from the arena walls.OFA: Mice were allowed 10 min to explore a white Plexiglas arena  under similar testing conditions to OFA and activity was analyzed with Ethovision XT as previously described .MWM: Mice were trained over 8 days as previously described  to locatFear conditioning and extinction: Mice were trained on day one with two pairings of a tone  and foot-shock  presentation, and freezing behavior was measured to assess fear acquisition. On day 2, contextual and cued long-term memory (LTM) were assessed by re-exposing mice to the training context  or to the CS in a novel context, respectively, with no US. Order of testing contextual and cued LTM with 1 hr between tests for each mouse was randomly counterbalanced among animals. Memory was measured as percent time spent freezing during the 5 min contextual test or during the two 30 s CS presentations given 1 min apart in the cued test. On day 7, mice received extinction training in the cued LTM testing environment over three consecutive days with a 35 min session each day consisting of 20 (30 s) CS exposures at varying intervals. On day 10, extinction LTM was assessed like cued LTM in a novel context. On day 20, renewal of conditioned fear was performed by re-exposing mice to the CS in the original training context using the same protocol as for training except no US was delivered. Baseline freezing was also monitored prior to the first CS presentation in any session. Context novelty was generated using alternate lighting (red light), walls and flooring (changeable acrylic inserts with different display patterns or textures), and odorants (vanilla or lavender) compared to the training environment.Akt1 KO mice were administered AAV (1 µL at 2.5e12 GC/mL over 10 min) to express either human AKT1-GFP  or Cre-GFP  under the human synapsin I promoter (hSyn) in each hemisphere. WT mice received only AAV-hSyn::Cre-GFP injections. Injectors were removed after an additional 10 min post-AAV infusion. Following standard post-operative care, mice remained in their home cages for 21 d post-surgery to allow AAV expression before fear memory and extinction testing. Following behavior tests, the brains from these mice were fixed and sectioned for microscopy using similar procedures described previously  for bilateral injections of recombinant AAV vectors into the prefrontal cortex (PFC) at Bregma coordinates 1.98 mm anterioposterior, ±0.5 mm mediolateral, and −2.35 mm dorsoventral. eviously  to confiBrain tissues were isolated from a subset of mice after completion of behavioral testing  and blotFluorescent immunostaining of fixed brain sections from transcardially perfused mice was performed using similar procedures described previously  to visuaAkt isoform mutants were maintained in separate lines and were not tested together, so Akt isoform experimental groups were compared only with their respective WT littermates and not analyzed together. Because male and female mice were tested separately, we also did not combine sexes for statistical analyses. However, we have included two-way ANOVA with sex and genotype as independent variables for each Akt mutant line . Data were analyzed for each sex by Student’s t-test, one-way analysis of variance (ANOVA) or one-way repeated-measures ANOVA where appropriate, with genotype as the between-subjects factor . Homogenant line  showing ant line . All beh In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:The role of molecular isoforms, highly similar molecules, in behaviors relevant to psychiatric disorders are not well elucidated. Using a series of genetic and behavioral approaches this research uncovers a new role for sex-specific contributions of molecular isoforms in distinct behaviors related to memory, anxiety, or fear. These findings have implications for understanding molecular mechanisms relevant to biological sex that occur in neuropsychiatric related behaviors.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Kate Wassum as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Isoform-specific roles for AKT in affective behavior, spatial memory, and extinction related to psychiatric disorders" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife'. Please let us know if you would like to pursue this option. As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv  along with this decision letter and a formal designation that the manuscript is 'in revision at Summary:This study examines AKT isoform-dependent effects on behaviors that are relevant to complex brain diseases. The study reveals differential selection of AKT1, AKT2, and AKT3 isoforms as a novel mechanism underlying anxiety, memory formation, and extinction. The authors further demonstrate that different AKT isoforms can compensate for each other, partially explaining the presence of very mild or null phenotypes. Overall, the manuscript expands upon the current limited knowledge of AKT signaling in maladaptive behaviors.Essential revisions:1) A concern with this study is that the results do not present a coherent conclusion, thus, it is not clear what is learned from this study. Please reshape the Introduction to clearly motivate the rationale to study these specific isoforms of AKT with genetic deletion, and what relevance this has for specific psychiatric disease and why the behavioral test performed are relevant. Additional comments below should also be integrated into this revised Introduction.2) In the Introduction, and some parts of the Discussion, the authors indicate that AKT is associated with MDD, and that antidepressant medications, among other psychotropic agents, modify AKT signaling. Yet, the authors provide only one reference that supports such statement for antipsychotics only . Please reference other sources to fully support such statements across the paper, particularly for antidepressants in human subjects .3) The NIH released a statement on the use of animals for research on psychiatric illness (https://grants.nih.gov/grants/guide/notice-files/NOT-MH-19-053.html). The statement emphasizes the importance of wording (including "of" versus "for") in descriptions and interpretations of the types of studies described in the present article. It would seem critical to ensure that the conceptualizations of genetically altered mice, and the behaviors they exhibit when compared to wildtypes, are made in the context of "modeling" endpoints that are suggestive of symptoms that underlie human psychiatric illnesses. Thus, it is imperative that the assertions in the present article are consistent with this NIH statement. As one example, the insinuation that the behaviors described  can accurately assess neuropsychiatric behavior in a mouse  may be viewed as increasingly unacceptable. Therefore, please revise to describe these tests in terms of their objective endpoints.4) Following the previous point, at the end of the Introduction, the authors indicate that their "findings have significant implications for diagnoses and therapies." Please describe how this statement is consistent with the NIH considerations regarding the use of animal neurobehavioral approaches in basic studies.5) One of prominent phenotypes in the study is a male-specific enhanced fear extinction in AKT1 KO. This can also be reversed by expressing AKT1 in the PFC. However, one of the main brain parts responsible for fear extinction is the amygdala and there are studies showing the connection between the PFC and amygdala may also play role in fear extinction. In addition, in Figure 6, conditional KO of AKT1 show normal extinction, which is possibly due to the fact that the cre line used in the study has not reported to have expression in the amygdala. This suggests AKT1 in the amygdala plays a role in fear extinction. Please examine whether AKT1 expression in the amygdala of KO mice can reverse fear extinction, or test whether specific loss of AKT1 in the amygdala  has any effects on behavior.6) The authors inject AKT1 into the Prelimbic cortex (PL), which is sufficient to reverse altered fear extinction in KO mice. Importantly, several studies show that the connection between the amygdala and PL mediates fear acquisition and retrieval while the link between the amygdala and infralimbic cortex (IL) are thought to mediate fear extinction . Please explain how AKT1 expression in the PL can reverse fear extinction or examine the effects of AKT1 expression in the IL on fear extinction in KO animals.Please also include a representative example of expression with sufficient display of anatomical landmarks to indicate that expression was restricted to the PL, as well as a schematic of expression spread overlaid for each subject so readers can see the extent of expression for the entire cohort.7) Across the manuscript, the authors indicate that their findings are "sex-dependent." This is technically inaccurate, given that sex was not incorporated as an independent variable in their statistical analyses. This is particularly problematic in the Discussion section of the paper, since the experimental design does not support such inferences. A similar problem is exhibited for the arguments made across the different AKT isoforms , since "isoform" was also not incorporated as an independent variable. The data should be re-analyzed using a 2-way ANOVA with "sex" and "AKT isoform" as sources of variance. Please also include a discussion on potential sex-dependent downstream players.8) In the Experimental Design and Statistical analysis section of the paper, the authors indicate that outliers were excluded from the study. The specific number of excluded animals, across each experiment, should be clearly indicated. Also, the current manuscript only includes group numbers. The total number (n) of animals per experimental group needs to be included . This is particularly important, given that the degrees of freedom change across experiments .Because the numbers of animals differ considerable between some of the groups, please also show that the data meet the assumption of homogeneity of variance for ANOVA.9) In Figure 6, please confirm loss of AKT1 expression by immunohistochemistry to examine the brain regions with AKT1 loss of expression.10) Given the complexity and length of the behavioral tests, it would be very useful if the authors included a schematic representation of the experimental timeline, depicting the multiple groups of AKT mutants, the rescue experiment, and the timeline of the behavioral tests.11) While the authors use a genetically targeted approach to study AKT isoforms, these molecules cross-talk with many signaling pathways. Therefore, it is very likely that multiple pathways are de-regulated by AKT KO. Assessing activation of other cross-talk signaling molecules  across the multiple AKT mutants (Western blotting) would provide a better understanding of the role that each AKT isoform plays in the AKT-dependent behavioral modifications.12) Some of the AKT KO mouse lines have previously been shown to be smaller and display growth deficiency . Are the same lines used in the current study. Are the mice smaller? Did the authors check the general health state of the mice? It is suggested is to add a short paragraph in the manuscript that clarifies this point.eLife. Your revised article has been evaluated by Kate Wassum (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled "Isoform-specific roles for AKT in affective behavior, spatial memory, and extinction related to psychiatric disorders" for further consideration by The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:1) The authors conclude that AKT1 in PFC interneurons is important for fear extinction. They showed that virally expressed AKT1 in the PFC reversed enhanced fear extinction in KO mice . However, the study lacks confirmation of AKT1 expression in interneurons. The authors are able to show double labeling of PV and AKT1 in the PFC in Figure 6—figure supplement 1. Thus, the authors should be able to perform similar labeling to demonstrate that virally expressed AKT1 is present in PFC interneurons in Figure 5C to confirm that AKT1 is indeed expressed in PFC interneurons of KO mice.2) Describing sex as an "experimental variable" throughout the manuscripts is inaccurate. The authors are to be commended for including both males and females in their work, and indeed, the inclusion of both sexes is extremely important. But not including sex as an independent variable in their statistical analyses (ANOVAs), precludes the authors from suggesting that sex was an experimental variable . There is agreement with the justification provided by the authors for not including sex or isoform as a variable in their statistical approaches. For this reason, it is encouraged that the authors remove any statement indicating that their findings are isoform or sex "dependent", and simply rephrase them as sex and isoform-"specific" (because data is compared to a same sex control). Essential revisions:1) A concern with this study is that the results do not present a coherent conclusion, thus, it is not clear what is learned from this study. Please reshape the Introduction to clearly motivate the rationale to study these specific isoforms of AKT with genetic deletion, and what relevance this has for specific psychiatric disease and why the behavioral test performed are relevant. Additional comments below should also be integrated into this revised Introduction.Akt isoform removal and viral-mediated gene expression to extend our results beyond null characterization and feel that these studies are very informative above and beyond what is known about AKT function in the context of behavior and how these behaviors may relate to human neuropsychiatric disorder.To address the reviewers’ concerns on this important point we have revised the Introduction as well as the Significance Statement to reflect more clearly the goals and knowledge gaps that motivated these studies. AKT has been implicated in neuropsychiatric disorders from human studies but it is a central kinase in many cellular signaling pathways, so understanding the specificity of AKT activity will be important for improving diagnosis and therapeutic interventions. Our study investigated the isoform- and sex-specific functions of AKT in mouse behaviors with underlying neural circuits and activity that may model brain processes impacted in neuropsychiatric disorders. The use of genetic deletion is a standard approach for understanding the contributions of a gene toward a process of interest. While there are other approaches , these approaches are not currently available for mouse model work without additional reagent generation. We did use the approach of conditional cell type-specific 2) In the Introduction, and some parts of the Discussion, the authors indicate that AKT is associated with MDD, and that antidepressant medications, among other psychotropic agents, modify AKT signaling. Yet, the authors provide only one reference that supports such statement for antipsychotics only . Please reference other sources to fully support such statements across the paper, particularly for antidepressants in human subjects .As suggested by the reviewers, we have included more references for AKT’s involvement in psychotropic agents, including antidepressants. These additional references add to the growing significance of the AKT signaling pathway in psychiatric disorders.3) The NIH released a statement on the use of animals for research on psychiatric illness (https://grants.nih.gov/grants/guide/notice-files/NOT-MH-19-053.html). The statement emphasizes the importance of wording (including "of" versus "for") in descriptions and interpretations of the types of studies described in the present article. It would seem critical to ensure that the conceptualizations of genetically altered mice, and the behaviors they exhibit when compared to wildtypes, are made in the context of "modeling" endpoints that are suggestive of symptoms that underlie human psychiatric illnesses. Thus, it is imperative that the assertions in the present article are consistent with this NIH statement. As one example, the insinuation that the behaviors described  can accurately assess neuropsychiatric behavior in a mouse  may be viewed as increasingly unacceptable. Therefore, please revise to describe these tests in terms of their objective endpoints.We understand that the behavioral measures we assessed in mice are endpoints which serve as models for examining effects on neural activity relevant to brain functions affected in psychiatric disorders and apologize for inadvertently using language that was not responsive to the NIH statement and guidance. We have revised the Introduction and wording in other places of the manuscript to be more objective and amenable to NOT-MH-19-053. The goals of our research were to identify behaviors and neurobiological processes impacted by AKT isoform activity and to examine them in the context of potential sex differences, which had been largely overlooked in previous preclinical studies. This has been more clearly delineated in the text.4) Following the previous point, at the end of the Introduction, the authors indicate that their "findings have significant implications for diagnoses and therapies." Please describe how this statement is consistent with the NIH considerations regarding the use of animal neurobehavioral approaches in basic studies.We have modified the Introduction to better fit the guidelines outlined in NOT-MH-19-053. In accordance with this notice, our study highlights the findings in the context of behavioral assays as readouts “for” brain function that may model neurobiological processes involved in psychiatric disorders. We delineate the contributions of AKT isoforms to specific brain processes (e.g. hippocampus-dependent long-term memory formation) and identify sex-dependent differences in the requirements for AKT activity.5) One of prominent phenotypes in the study is a male-specific enhanced fear extinction in AKT1 KO. This can also be reversed by expressing AKT1 in the PFC. However, one of the main brain parts responsible for fear extinction is the amygdala and there are studies showing the connection between the PFC and amygdala may also play role in fear extinction. In addition, in Figure 6, conditional KO of AKT1 show normal extinction, which is possibly due to the fact that the cre line used in the study has not reported to have expression in the amygdala. This suggests AKT1 in the amygdala plays a role in fear extinction. Please examine whether AKT1 expression in the amygdala of KO mice can reverse fear extinction, or test whether specific loss of AKT1 in the amygdala  has any effects on behavior.Akt1 KO (cAkt1 KO) mice driven by the T29-1 line . We show further that this line expresses in excitatory neurons and not interneurons of the amygdala and PFC . Taken together with our finding that neuronal AKT1 expression in the PFC of Akt1 KO mice reverses their fear extinction phenotype, these data suggest that interneuronal AKT1 in the PFC plays a critical role in fear extinction. Therefore, cAkt1 KO mice showed normal extinction possibly because AKT1 expression remained intact in interneurons of the PFC. We do not dispute the important role of the amygdala in extinction, but our data indicate that AKT1 is not required in excitatory neuronal populations of the amygdala or PFC for the normal display of extinction. To more thoroughly test this idea, we plan to generate interneuron-specific Akt1 KO mice and examine fear extinction in these animals for future studies. We have included this discussion in the revised manuscript. Based on these data, we did not pursue behavioral studies to test the role of AKT1 specifically in the amygdala, although we agree that it would be interesting to examine as part of a larger future study of brain region-specific AKT1 functions.The particular Cre line used for this study, T29-1 , does in fact express quite well in the amygdala. In a previous report, we confirmed its expression in the amygdala . To provide additional support, we have included immunoblotting and histological data confirming reduced AKT1 expression in the amygdala as well as in the PFC of conditional 6) The authors inject AKT1 into the Prelimbic cortex (PL), which is sufficient to reverse altered fear extinction in KO mice. Importantly, several studies show that the connection between the amygdala and PL mediates fear acquisition and retrieval while the link between the amygdala and infralimbic cortex (IL) are thought to mediate fear extinction . Please explain how AKT1 expression in the PL can reverse fear extinction or examine the effects of AKT1 expression in the IL on fear extinction in KO animals.Please also include a representative example of expression with sufficient display of anatomical landmarks to indicate that expression was restricted to the PL, as well as a schematic of expression spread overlaid for each subject so readers can see the extent of expression for the entire cohort.Akt1 KO mice is that AKT1 activity is normally required in the mPFC to limit extinction learning. While examining the role of AKT1 activity in specific PFC regions would be an interesting line of inquiry, based on our results from forebrain excitatory neuron-specific removal of AKT1, we posit that the AKT1 activity utilized during extinction learning is primarily required in interneuronal populations of the PFC . The AAV-AKT1 construct we used expresses in cells responsive to the synapsin promoter, so it does not resolve AKT1 activity in excitatory or inhibitory neurons . Therefore, as mentioned for point #5, our future efforts will directly test the idea that interneuronal AKT1 is responsible for normal extinction behavior using genetic approaches to eliminate AKT1 and AAV constructs designed to limit AKT1 expression to interneurons.We apologize that the methods for viral-mediated AKT1 expression was not clearly described in the original submission. Although we targeted the PL region of the medial prefrontal cortex (mPFC), it was not our intention to imply that viral expression was limited to the PL. At the reviewers’ suggestion, we provide a representative example of AAV expression in the PFC along with a schematic of the expression spread overlaid for available subjects, which we have incorporated into Figure 5. While AAV expression was visually confirmed for every subject, we had only comprehensively imaged a few representative samples. As these experiments were performed over the course of a couple years, the original slides for confirming expression were no longer usable for imaging. We prepared new slides but only some subjects had brain sections with PFC available. To construct the schematic, we compiled images from this subset, which consists of samples from each group. The revised figure shows that both the PL and IL exhibit AAV expression . We do not distinguish between the requirements for AKT1 activity in PL or IL in this study. Instead, we elected for a more general approach, indeed given some conflicting reports regarding the requirement for the PL and IL in fear extinction processes . Our simplest interpretation of the results from restoring AKT1 expression in the mPFC of 7) Across the manuscript, the authors indicate that their findings are "sex-dependent." This is technically inaccurate, given that sex was not incorporated as an independent variable in their statistical analyses. This is particularly problematic in the Discussion section of the paper, since the experimental design does not support such inferences. A similar problem is exhibited for the arguments made across the different AKT isoforms , since "isoform" was also not incorporated as an independent variable. The data should be re-analyzed using a 2-way ANOVA with "sex" and "AKT isoform" as sources of variance. Please also include a discussion on potential sex-dependent downstream players.Akt isoform differences. Below we present our rationale for the way we conducted the analyses.This is a very important point, and we carefully considered the implications of our research design and interpretations to address this critique. It appears to us that many labs employ a variety of statistical approaches, some applied correctly, some applied incorrectly. It is also the case that there are often multiple statistical approaches that could be used, and if the “take-home” message doesn’t change it can simply be a matter of preference. The two main issues raised by reviewers for this manuscript focus on how to handle possible sex differences and whether to examine possible 1) Sex differences: We do not share the reviewers’ interpretation about the use of “sex-dependent.” Behavioral effects observed in one sex but not another is sex-dependent. However, we understand the concept of including sex as a variable in our statistical analyses. In a previous submission to another journal that included only a subset of these data, we did in fact prepare our manuscript with two-way ANOVA analyses (genotype and sex) but received criticisms about that approach. In that case, the reviewers requested post-hoc analyses, but post-hoc tests are not appropriate when there are only two levels for each variable (e.g. genotype can be only KO/WT and sex can be only M/F). Therefore, we consulted with local biostatistical experts (Dr. Luke Evans and Dr. Marissa Ehringer) and revised our figures based on their directions.a priori knowledge of such sex differences , analyses are frequently performed separately by sex to simplify the presentation and interpretation of results. It is not uncommon for researchers to actually submit two different papers, one for each sex. In our case, the behaviors we examined are also well-known to show reproducible sex differences in WT (C57BL/6) strains . Thus, to avoid complicating the current submission with results of behavioral differences inherent to sex alone, which are already known, and because males and females within each genotype set were tested separately, we opted to perform within-sex t-tests . This analysis facilitated presentation of our results for easy comparison between WT and KO genotypes within either male or female groups. It allowed us to show data normalized to WT controls within each sex in order to focus on the effect of Akt isoform manipulation, which was the primary goal of our study. At the reviewers’ suggestion, we also performed two-way ANOVA, and the overall “take-home” message does not change. This analysis is included in Table 4. If all reviewers and the editor strongly feel we should present the results as a two-way ANOVA, we can change our figures. Based on our previous submission experience, we expect this might cause more confusion than the simpler t-test, which we believe is justified based on previously known sex differences and the experimental design.Dr. Ehringer deals with this sex effect issue on a regular basis because there are well-known sex differences in both rodents and humans for alcohol-related behaviors, which is a major area of her research. Given the Akt isoform as an independent variable for analyses is not appropriate for four reasons. First, the individual Akt mutant lines are bred and maintained separately. They cannot be reasonably grouped all together because they are maintained on different backgrounds and not simply interchangeable. In order to analyze the data as proposed , all the mutant Akt alleles would have to be maintained in the same strain. This was not done for practical reasons; it would require thousands of progeny from Akt1+/-Akt2+/-Akt3+/- breeder mice to complete these studies in this way and only 1.6% of mice derived from such a cross would be either pure WT or KO for each isoform group. Second, although all the mice are maintained on a C57Bl/6 background, the different Akt lines are not isogenic. We regularly introduce new C57Bl/6 mice into all our strains to prevent the accumulation of genetic modifiers. Despite this precaution, “WT” mice from each strain demonstrate slight performance differences in some tasks compared with “WT” mice from other Akt isoform strains. There are many possible reasons for this  but those factors are largely beyond our control. Therefore, it would not be scientifically appropriate to compare KO mice to “WT” mice from a different line or to create a master “WT” group by simply averaging the performance of WT mice from all lines. Third, we used littermates obtained from breeding the individual Akt lines for each experimental group, a practice that is supported by previous studies . This important element of rigorous experimental design would be lost if we were to combine across all isoform groups. Finally, the experiments in this study were not designed or performed with the goal of comparing each Akt mutant line to one other. Combining all the mutants for simultaneous comparison creates an insurmountable multiple comparison problem that would necessitate testing many more animals, as the power analyses used to design our experiments were based on separate isoform testing. Another way to view the situation is that because the experiments were performed with only one mutant at a time, it would have been reasonable and appropriate to prepare three different manuscripts, one for each isoform, which would be analyzed independently.2) AKT isoform comparisons: We believe including At the reviewers’ request, we also included in the revised manuscript a discussion on potential sex-dependent downstream players, which incorporates the findings from our experiments to address point #11 below.8) In the Experimental Design and Statistical analysis section of the paper, the authors indicate that outliers were excluded from the study. The specific number of excluded animals, across each experiment, should be clearly indicated. Also, the current manuscript only includes group numbers. The total number (n) of animals per experimental group needs to be included . This is particularly important, given that the degrees of freedom change across experiments .Because the numbers of animals differ considerable between some of the groups, please also show that the data meet the assumption of homogeneity of variance for ANOVA.We apologize that presentation of the experimental details for our statistical analysis was confusing in the original submission. We have now added summary tables with the number of animals per experimental group , number of excluded animals and reason for exclusion, and homogeneity of variance test results across each experiment (Tables 2-4).9) In Figure 6, please confirm loss of AKT1 expression by immunohistochemistry to examine the brain regions with AKT1 loss of expression.Akt1 removal under the T29-1 Camk2α-Cre driver reduced AKT1 expression in the hippocampus . Complementing this report, we now demonstrate using western blotting and immunohistochemistry that AKT1 expression is also reduced in the amygdala and PFC. These data have been added as a supplemental figure .We previously confirmed that conditional 10) Given the complexity and length of the behavioral tests, it would be very useful if the authors included a schematic representation of the experimental timeline, depicting the multiple groups of AKT mutants, the rescue experiment, and the timeline of the behavioral tests.We thank the reviewers for this very useful suggestion and have amended our Materials and methods section to include a flowchart for the behavioral assays .11) While the authors use a genetically targeted approach to study AKT isoforms, these molecules cross-talk with many signaling pathways. Therefore, it is very likely that multiple pathways are de-regulated by AKT KO. Assessing activation of other cross-talk signaling molecules  across the multiple AKT mutants (Western blotting) would provide a better understanding of the role that each AKT isoform plays in the AKT-dependent behavioral modifications.Akt isoform mutants of both sexes. These data are now included as Figure 9 and associated supplemental figures. While we did not identify many differences in this analysis, we did find some exciting isoform- and sex-dependent effects on GSK3β phosphorylation. We hope to investigate other potential signaling differences that may mediate the behavioral effects of Akt isoform deficiency using a more comprehensive approach, such as mass spectrometry, in a follow-up study.We agree that loss of AKT likely influences many different signaling pathways and that these may be involved in the behavioral effects we observed. Following the reviewers’ suggestion, we performed western blot analysis of PDK1, GSK3β, and ERK activation to assess candidate signaling upstream, downstream, and parallel of AKT, respectively, in brain tissue from 12) Some of the AKT KO mouse lines have previously been shown to be smaller and display growth deficiency . Are the same lines used in the current study. Are the mice smaller? Did the authors check the general health state of the mice? It is suggested is to add a short paragraph in the manuscript that clarifies this point.Akt1 and Akt2 KO lines from JAX, donated by Morris Birnbaum. Our Akt3 KO line was also obtained from the Birnbaum Lab by direct request and is not the same strain described in Dummler et al. 2006. This information has been added to the Key Resources Table. In our hands, both Akt1 and Akt3 KO mice show significantly reduced sizes compared with their WT littermates. The size reduction is slight in Akt1 KO mice and more pronounced in Akt3 KO mice, consistent with previous reports ; Easton et al., 2005). There are conflicting reports of Akt2 KO mice having a reduced weight ; Garofalo et al., 2003). In our hands, Akt2 KO mice trend towards a reduced size but the difference is not significant. Akt2 KO mice do develop insulinemia that becomes progressively worse with age . Most glucose-related phenotypes in Akt2 KO mice are uncovered following fasting or with aging, but our mice have access to food ad libitum and were tested younger than 16 weeks, before insulin-related phenotypes become severe. Previous studies examined the general health of all these KO mice, and apart from size, found them generally indistinguishable from their WT littermates . In addition, we assessed sensory functions  and found no differences between KO and WT mice. Locomotor assessments, such as distance moved and swim velocity, were included in our behavioral assays to rule out locomotor defects as a potential confound for observed behavioral differences. None were identified. Some of these control data are included in the submitted figures . We have added a paragraph about general health to the Materials and methods section to make clear this point.We obtained our The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:1) The authors conclude that AKT1 in PFC interneurons is important for fear extinction. They showed that virally expressed AKT1 in the PFC reversed enhanced fear extinction in KO mice . However, the study lacks confirmation of AKT1 expression in interneurons. The authors are able to show double labeling of PV and AKT1 in the PFC in Figure 6—figure supplement 1. Thus, the authors should be able to perform similar labeling to demonstrate that virally expressed AKT1 is present in PFC interneurons in Figure 5C to confirm that AKT1 is indeed expressed in PFC interneurons of KO mice.Akt1 KO mice is present in interneurons with our AAV construct .As suggested, we now include images confirming that virally expressed AKT1 in the PFC of 2) Describing sex as an "experimental variable" throughout the manuscripts is inaccurate. The authors are to be commended for including both males and females in their work, and indeed, the inclusion of both sexes is extremely important. But not including sex as an independent variable in their statistical analyses (ANOVAs), precludes the authors from suggesting that sex was an experimental variable . There is agreement with the justification provided by the authors for not including sex or isoform as a variable in their statistical approaches. For this reason, it is encouraged that the authors remove any statement indicating that their findings are isoform or sex "dependent", and simply rephrase them as sex and isoform-"specific" (because data is compared to a same sex control).We have changed references of “dependent” to “specific” throughout the text.


Our DL model showed that the DLT–VO2 was confirmed to be significantly correlated with the VT–VO2 , and the mean difference was nonsignificant , which displayed strong agreements between the VT and the DLT. The DL algorithm using single-lead ECG data enabled accurate estimation of VT in patients with CVDs. The DL algorithm may be a novel way for estimating aerobic exercise threshold.Regular aerobic physical activity is of utmost importance in maintaining a good health status and preventing cardiovascular diseases (CVDs). Although cardiopulmonary exercise testing (CPX) is an essential examination for noninvasive estimation of ventilatory threshold (VT), defined as the clinically equivalent to aerobic exercise, its evaluation requires an expensive respiratory gas analyzer and expertize. To address these inconveniences, this study investigated the feasibility of a deep learning (DL) algorithm with single-lead electrocardiography (ECG) for estimating the aerobic exercise threshold. Two hundred sixty consecutive patients with CVDs who underwent CPX were analyzed. Single-lead ECG data were stored as time-series voltage data with a sampling rate of 1000 Hz. The data of preprocessed ECG and time point at VT calculated by respiratory gas analyzer were used to train a neural network. The trained model was applied on an independent test cohort, and the DL threshold  was calculated. We compared the correlation between oxygen uptake of the VT (VT–VO In contrast, an unexpected high-intensity exercise could be a cause of deterioration or hospitalization in patients with CVDs6; current clinical practice guidelines and expert statements recommend aerobic exercise for patients with CVDs8. Although cardiopulmonary exercise testing (CPX) is an essential examination for noninvasively detecting the ventilatory threshold (VT), that is, defined as clinically equivalent to aerobic exercise, its assessment requires an expensive respiratory gas analyzer and expertize. In this context, expansion of the exercise therapy with a simple, versatile methodology to facilitate its introduction and persistency is warranted to improve clinical outcomes of patients with CVDs.Adequate regular physical activity is paramount to maintaining good health9  in cardiovascular medicine. In addition, DL aids in the interpretation of clinically important findings from imaging data to support clinical judgment by physicians15. However, there is limited evidence for estimating the threshold of aerobic exercise with DL algorithms combined with neural networks. Herein, we aimed to investigate the feasibility of a DL algorithm with single-lead ECG during incremental exercise for estimating the aerobic exercise threshold in patients with CVDs.Advancement of high-performance computer technology and deep learning (DL) technology has enabled generation of models that accurately predict outcomes, detect diseases, and automatically classify or quantitate measurements from various modalities including electrophysiological and imaging dataFrom April 2014 to May 2019, 404 patients underwent CPX in Keio University Hospital , diabetes mellitus (18.1%), and dyslipidemia (40.4%). Medical history of CVD included chronic heart failure (46.5%), coronary artery disease (38.4%), pulmonary hypertension (15.8%), and arrhythmias (14.2%). The details of each medical history are provided in Supplementary Table 2) in the train and test cohort  was compared with the VT–VO2 manually detected from a respiratory gas analyzer during CPX. The relationship between the VT–VO2 and the DLT–VO2 of the train cohorts showed a satisfactory result  was nonsignificant  were >0.7 in all the subgroups, and there were no major differences among stratified characteristics .The correlation coefficient between the VT–VOIn the present study, we found that the DL algorithm constructed with neural networks from single-lead ECG data during exercise enabled estimation of the VT in patients with CVDs. This study is unique in that we focused on the fact that electrical activity of the heart is dynamic during exercise and that the changes derived from hidden big data might resemble the feature of the VT.16 reported that deep neural networks could have application in the context of CPX for detecting cardiovascular outcome in patients with heart failure. Hearn et al.17 also described that harnessing neural networks with CPX data improved the prognostication of outcomes compared with the conventional prognostic method in patients with heart failure. These two studies implemented DL for the detection of prognosis. The strong point of our study is that our DL algorithm could recognize hidden features from single-lead ECG and estimate the VT, which was a clinically essential parameter for determining the level of exercise.DL is bringing a paradigm shift to healthcare, which is powered by increasing availability of healthcare data and rapid progress of analytic techniques. In recent years, the application of DL to cardiovascular medicine has advanced rapidly. Especially since the learning method called neural network has spread, we have made remarkable progress in precision. In the area of cardiovascular medicine, there are many applications of DL using neural networks; however, few reports are available in the area of cardiac rehabilitation. Myers et al.18. The two-dimensional convolution network is useful in extracting information from a still image19. This is done by abstracting local information within the special axis of the images. The 1D-conv network does the similar abstraction but on only a single axis; in our case, it was a time axis. The LSTM is an improved version of the recurrent neural network, which is effective in learning time-series data. However, this network expands the steps of back propagation as the length of the input data increases. This causes the problem of vanishing gradient and makes the training computationally expensive. The combination of 1D-conv and LSTM may have been powerful for dealing with long time-series data by allowing abstraction of complex ECG patterns by 1D-conv and reducing the complexity of the data that the LSTM should learn. However, in order to deal with a larger number of voltages recorded in a single dataset, our study modified the network to form a more complex structure but was based on the same principal of the previously reported combination. We applied an ECG data length of 30 s to run our network structure combining 1D-conv and LSTM. We did not compare the model performance using other ECG length. A systematic research of the best ECG length may have further improved the model. Our study again shows that the combination of 1D-conv and LSTM is a powerful tool for dealing with time-series data of voltage recordings from ECG.Our network structure combining one-dimensional convolution (1D-conv) and long–short term memory (LSTM) efficiently learned the complex time-series pattern of voltage in an ECG record and effectively estimated the time point of VT without the need for a respiratory gas analyzer. This combination of 1D-conv and LSTM has been shown to be effective in learning 12-lead ECG in our previous investigation20. Nevertheless, the application of cardiac rehabilitation for patients with CVDs is extremely low, especially in the outpatient setting23. Some factors contribute to such situations: complexity of CPX, time conflicts, and patients’ disinterest and uncertainty about the management of aerobic exercise in daily life24. Alternative methods are needed to facilitate the estimation of aerobic exercise thresholds and expand exercise therapy to the outpatient setting. We have previously demonstrated that a real-time evaluation of the HR variability (HRV) with single-lead ECG during CPX could be helpful for detecting the aerobic exercise threshold25. The study targeted patients who had myocardial infarction and sinus rhythm on ECG but not arrhythmia, such as AF, because the HRV analysis is not applicable in patients with irregular R–R intervals on ECG. In contrast, the method combined with the DL algorithm in the present study could be expanded to a wide range of CVDs patients, including those with AF during CPX. Further, if the algorithm of this study is mounted on wearable devices that can record ECGs, it can improve the persistence of cardiac rehabilitation programs in outpatients and relocate their bases from hospitals to other institutions, such as commercial fitness clubs or even patients’ homes.Cardiac rehabilitation defined by multidisciplinary professionals plays an important role in the disease management program for patients with CVDs, leading to improved exercise tolerance and quality of life, and reduced hospitalization227. We also tested model excluding ECG data by training the same DL architecture with dummy ECG  to demonstrate the validity of the present study. In the model, there was also a correlation between DLT–VO2 and VT–VO2 of r = 0.771 . These results suggest that ECG plays a crucial role in improving the accuracy of our model to achieve a good estimation for the VT. Third, the model requires the exercise time as an input. Therefore, it is not applicable to patients who cannot complete the CPX until exhaustion. Fourth, we could estimate the VT using DL including cardiac rhythm abnormalities , and there was no significant deference in the DL model regardless of whether patients had AF ; therefore, further studies should be performed to assess the efficacy of our model in such patients. Finally, if used practically, the DL algorithm can estimate HR and work rate . At first, the patients rested for 2 min on the ergometer until their heart rate (HR) and respiratory condition slowed down. Following a 2-min rest (rest phase), the patients performed a 2-min warm-up pedaling at 0 W (warm-up phase). The intensity was increased with a RAMP protocol ergometer (10–15 W/min), depending on the exercise capacities of each patient (exercise phase). The patients exercised with a progressive intensity until they could no longer maintain the pedaling rate . After the exercise tests were terminated, the patients were instructed to stop pedaling and to stay on the ergometer for 3 min (recovery phase). The blood pressure was measured every minute with an indirect automatic manometer. Single-lead and 12-lead electrocardiograms were continuously recorded during whole test from the beginning of the rest phase to the end of the recovery phase.Vmax; Nihon Kohden, Tokyo, Japan). The respiratory gas exchange, including the ventilation, VO2, and carbon dioxide production, were monitored continuously and measured using a 30-s average. This system was subjected to a 3-way calibration process, involving a flow volume sensor, gas analyzer, and delay time calibration. VT was determined conventionally using the procedures described by Gaskill et al. 28. The peak VO2 was calculated as the average oxygen consumption during the last 30 s of exercise. The ventilation/carbon dioxide (ventilator efficiency) slope (VE–VCO2 slope) was based on data from the onset of exercise to the respiratory compensation point, and it was obtained by performing a linear regression analysis of the data acquired throughout the entire period of exercise30. The respiration quotients at VT and peak exercise were measured.The expired gas flows were measured using a breath-by-breath automated system  with TensorFlow31 as backend. The neural network was trained using the back-propagation supervised training algorithm. The loss function of binary cross entropy was minimized using the RMSprop optimizer . The network was trained for 60 epochs, and the model that performed best with the validation cohort was selected as the final model , 72 (27.7%), and 91 35.0%) patients were randomly assigned to derivation, validation, and test cohorts, respectively. A combination of the derivation and validation cohorts were grouped with the train cohort , left ventricular ejection fraction, rhythm of AF during CPX, and prescription of a β blocker, and estimated the relationship between the subgroups in the test cohort. Correlation coefficients between the full model and the model without ECG were compared using the correlation coefficient difference test. All probability values were two-tailed, and P values < 0.05 were considered to be statistically significant. All statistical analyses were performed with SPSS version 25.0 software .The results are represented as the mean ± standard deviation for continuous variables and as a percentage for categorical variables, as appropriate. The relationships between the studied methods of the VT and the DLT as derivation, validation, and test cohorts were investigated by using the Pearson correlation coefficient test. The Bland–Altman technique was applied to verify the similarities between the different methods (VT and DLT)The study protocol was approved by the Institutional Review Board of Keio University School of Medicine (permission number: 2014023) and conducted in accordance with the Declaration of Helsinki. All patients provided written informed consent.Further information on research design is available in the Nature Research Reporting Summary linked to this article.Supplementary InformationReporting Summary