As part of the Global Action Plan against antimicrobial resistance (AMR), countries are required to generate local evidence to inform context-specific implementation of national action plans against AMR (NAPAR). We aimed to evaluate the knowledge, attitude, and practice (KAP) regarding antibiotic prescriptions (APR) and AMR among physicians in tertiary hospitals in Nigeria, and to determine predictors of KAP of APR and AMR.In this cross-sectional study, we enrolled physicians practicing in tertiary hospitals from all six geopolitical zones of Nigeria. Implementation of an antimicrobial stewardship programmes (ASP) by each selected hospital were assessed using a 12 item ASP checklist. We used a structured self-administered questionnaire to assess the KAP of APR and AMR. Frequency of prescriptions of 18 different antibiotics in the prior 6 months was assessed using a Likert’s scale. KAP and prescription (Pr) scores were classified as good (score ≥ 80%) or average/poor (score < 80%). Independent predictors of good knowledge, attitude, and practice (KAPPr) were ascertained using an unconditional logistic regression model.A total of 1324 physicians out of 1778 (74% response rate) practicing in 12 tertiary hospitals in 11 states across all six geopolitical zones participated in the study. None of the participating hospitals had a formal ASP programme and majority did not implement antimicrobial stewardship strategies. The median KAPPr scores were 71.1%, 77%, 75% and 53.3%, for the knowledge, attitude, practice, and prescription components, respectively. Only 22.3%, 40.3%, 31.6% and 31.7% of study respondents had good KAPPr, respectively. All respondents had prescribed one or more antibiotics in the prior 6 months, mostly Amoxicillin-clavulanate (98%), fluoroquinolones (97%), and ceftriaxone (96.8%). About 68% of respondents had prescribed antibiotics from the World Health Organization reserve group. Prior AMR training, professional rank, department, and hospital of practice were independently associated with good KAPPr.Our study suggests gaps in knowledge and attitude of APR and AMR with inappropriate prescriptions of antibiotics among physicians practicing in tertiary hospitals in Nigeria. Nigeria’s NAPAR should also target establishment and improvement of ASP in hospitals and address institutional, educational, and professional factors that may influence emergence of AMR in Nigeria. The discovery of antibiotics has completely revolutionized medical practice and led to a decrease in the morbidity and mortality due to infectious diseases across the globe . HoweverWhile the threat of antimicrobial resistance (AMR) remains a growing global challenge, developing countries in Asia and Africa are at greatest risk. It is estimated that by 2050, if nothing is done to halt the increasing trend of AMR, about 10 million people will die from AMR globally, including about 4 million people each from Asia and Africa . The AccInfectious diseases remain the commonest cause of disease morbidity and mortality in Nigeria . FollowiSeveral studies within and outside Nigeria have shown that AMRO occur commonly among patients admitted in intensive care units, paediatric units and other specialist units often situated within tertiary hospitals , 13. TheImplementation of antimicrobial stewardship programmes (ASP) lies at the core of optimizing appropriate use of antibiotics in healthcare facilities , 16. AnyPrevious studies have shown that only 13–35% of tertiary hospitals in Nigeria had a formal ASP and majority did not implement most ASM-related strategies , 18. OthWe aimed to determine the KAP of APR and AMR, and identify sociodemographic, educational, institutional, and professional factors that may be associated with good KAP of APR and AMR among physicians in tertiary hospitals across all six geopolitical zones of Nigeria. Our overarching goal was to generate valuable sentinel national data to inform implementation of the country’s NAPAR.This cross-sectional study was conducted between October 2018 and February 2019 among physicians practicing in public tertiary hospitals in Nigeria.Nigeria runs a federal system of government consisting of a Federal Government, 36 semi-autonomous State Governments, the Federal Capital Territory (FCT) and 774 Local Government Areas. The states and FCT are grouped into six geo-political zones: South-South, South-East, South-West, North-East, North-West, and North Central. There are 101 public tertiary health facilities across all the 36 states and the FCT of Nigeria . These pWe selected 12 tertiary hospitals across the country by convenience sampling and included at least one hospital from each of the six geopolitical zones of the country. In a bid to generate representative data, and allow for appropriate sub-group analysis, we targeted at least 40% of physicians working in each hospital. We then adopted a purposive sampling technique to guarantee inclusion of physicians of all professional ranks and departments in each of the selected hospitals. There were 4446 physicians in all 12 hospitals at the time of the study, and 1778 (40%) were eligible to be enrolled for the study.A structured standardized self-administered pretested questionnaire was distributed to the study participants by trained research assistants who ensured that all completed questionnaires were returned the same day.The questionnaire consisted of questions divided into four sections: (1) Implementation of antibiotic stewardship programmes (ASP) in selected hospitals and prior training on APR and AMR (2) Demographic and occupational characteristics of study participants; (3) Knowledge, attitude and practice of antibiotic prescriptions and resistance. (4) Frequency of prescriptions of 18 different antibiotics in the 6 months prior to the study.The questionnaire was developed after reviewing qualitative and quantitative literatures for relevant items , 23, incA checklist consisting of 12 items assessed compliance with elements of ASP , knowledge of rational use of antibiotics (eight questions), definition of AMR (one question) and knowledge of causes of antimicrobial resistance (11 questions). Correct responses were scored 1 while each wrong or do not know answer was scored 0. The overall knowledge score ranged from 0 to 28.Attitude was assessed by a set of 15 positive and negative attitude questions using a five-point Likert’s scale. The scoring system used for positive attitude was: 5 = strongly agree with the statement, 4 = agree, 3 = neutral, 2 = disagree and 1 = strongly disagree with the statement. The scores were reversed for negative attitude questions. Overall attitude scores ranged from 15 to 75.Practice was assessed by a set of 13 positive and negative practice questions using a five-point Likert’s scale. The scoring system used for correct practice was: 5 = Always, 4 = Most Often, 3 = Sometimes, 2 = Rarely and 1 = Never. The scores were reversed for wrong practice questions. Overall practice scores ranged from 13 to 65.To determine the frequency of prescriptions of 18 different antibiotics, respondents were asked to indicate how many times they prescribed each antibiotic in the previous 6 months using a Likert’s scale of Never, Rarely, Sometimes, Most Often, Always, scored 1, 2, 3, 4 and 5 respectively. Overall prescription scores ranged from 18 to 90.We calculated percentage scores for each ASP, KAP and prescription score. The 18 antibiotics were further classified into the WHO AWaRe categories . At the Using modified Bloom’s cut-off point , the perThe collected data were checked for completeness and errors before analysis. Incomplete questionnaires were excluded and counted as a non-response. All completed questionnaires were then entered into Microsoft Excel and exported to SPSS version 20 for analysis.p value < 0.05 (two-sided) was considered statistically significant.Study variables were summarized using frequencies, proportions, medians, and interquartile range-IQR . Spearman rho’s correlation coefficient was used to describe the strength and direction of the relationship among KAPPr scores. Correlations were interpreted using the following criteria: 0–0.25 = weak correlation, 0.25–0.5 = fair correlation, 0.5–0.75 = good correlation and greater than 0.75 = excellent correlation. A chi-squared test was used for bivariate analysis. For multivariate analysis, KAPPr scores were grouped into two: Good KAPPr (scores ≥ 80%) and Average/Poor KAPPr (scores < 80%). An unconditional binary logistic regression was used to determine independent variables associated with good KAPPr. Adjusted odds ratio with 95% confidence level were computed, and All potential participants who agreed to participate provided written consent before completion of questionnaire. The questionnaire was anonymized, and confidentiality of information obtained was maintained. Ethical approval for the study was obtained from National Health Research Ethics Committee (NHREC/01/01/2017).Of 1778 questionnaires, 1324 (74% response rate) were completed and returned from 11 states and 12 hospitals across all geopolitical zones of Nigeria  of 2 . None of the hospitals surveyed had a formal ASP or a policy on antibiotic restrictions  reported participating in a prior training on antibiotic use and resistance.The majority (> 80%) of study participants had heard of rational antibiotic prescription and AMR, but only 40.6% had heard of ASP Table . AwareneConcerning attitude toward antibiotic use and resistance, majority of participants agreed that AMR is a serious public health issue in Nigeria and worldwide and could be a problem for their hospital Table . HoweverTable All study participants (100%) had prescribed one or more antibiotics in the previous 6 months. A median prescription of 15 antibiotics IQR-13, 17) were prescribed per study participant in the previous 6 months. The prescription scores ranged from 20 to 90 with median (IQR) of 48 . Amoxicillin-clavulanate (98%), ciprofloxacin/ofloxacin (97%), ceftriaxone (96.8%) and metronidazole (96.5%) were the most frequently prescribed antibiotics  were more likely to have good KAPPr than those respondents from other hospitals Fig. . RespondTo the best of our knowledge, this is the first study from Nigeria assessing both implementation of ASM among tertiary hospitals and predictors of good KAP of APR and AMR among physicians practicing in all six geopolitical zones of Nigeria.Our results suggest significant gaps in the implementation of antimicrobial stewardship (AMS). None of the hospitals surveyed had a formal ASP program, while majority of participating hospitals were not routinely implementing one or more of the ASM-related interventions and there was poor awareness of ASM. Poor compliance with AMS was reported by another study from Nigeria where only six (35%) of 17 tertiary hospitals surveyed had a formal ASP and less than 23.5% of these hospitals implemented AMS-related interventions and policies . In lineOur study respondents had better knowledge of AMR (median score of 72.7%) than knowledge of rational APR (median score of 62.5%). Most respondents agreed that AMR is a serious problem in their various hospitals and Nigeria as a whole. However, over half of our respondents did not know that AMR could arise from antibiotic use in animal husbandry and farming. About 78% of farms in Nigeria are using antimicrobials for animal production, and 20% of these livestock have been shown to have multi-drug resistant isolates . Our resThe gap in knowledge of the link between poor practice of IPC and AMR as observed in our study may partly explain why 79% of respondents disagreed that routine hand washing could prevent AMR and 36% were neutral or disagreed that AMR could be transmitted from healthcare worker to patient. About 83% of physicians in another study from Nigeria identified poor infection control as a possible cause of emergence of AMR in hospital settings . HoweverThe median KAP scores of respondents in our study were 71%, 75% and 77% respectively. However, only 22.3%, 40.3%, and 31.6 had good KAP (defined as score of 80% or above) of APR and AMR, respectively. In a systematic review of KAP of APR and AMR among healthcare practitioners from developing countries involving 15 studies (none from Nigeria) an average of 80.9% of respondents correctly answered questions relating to APR, whereas only 39.6% were aware of the local resistance patterns in their health facilities . About 8Excessive and inappropriate prescription of antibiotics are established drivers of AMR , 36. OurMany studies from Nigeria have also reported overuse and irrational prescription of antibiotics among physicians, with prevalence of self-reported APR ranging from 26.8 to 97% , 20, 37 The determinants of inappropriate prescriptions of antibiotics have been reviewed by various authors , 43. A sEducational programmes have been shown to improve awareness and knowledge of AMR among healthcare workers, as well as foster appropriate prescription behaviour , 45. We Independent of other variables, more senior physicians (consultants and resident doctors) in our study had better knowledge and attitudes, and prescribed antibiotics less frequently than recently qualified medical interns. Medical interns have limited experience of clinical practice, and it is not surprising they had the lowest KAPPr scores in our study. Besides poor knowledge, the more frequent prescriptions of antibiotics among these interns compared to other physicians could be related to the hierarchical nature of medical practice in Nigeria where medical interns are in most cases required to write out prescriptions based on instructions of their supervising residents or consultants. The rotation of interns through all major medical specialties has been shown to influence their prescription practice as they are made to transcribe prescriptions from a wide variety of their supervisors in the various departments . StudiesHospital of practice was independently associated with good KAPPr among physicians in our study. Physicians practicing in hospitals located in the South-West region of the country generally had better KAPPr scores than those from other hospitals. Prescriptions of Reserve antibiotic (Cefepime) were more frequently observed among physicians practicing in northern Nigeria, as all three hospitals with over 80% of physicians with prior prescriptions of Cefepime were in northern Nigeria. Among five published PPS from Nigeria –41, 54, Variabilities between hospitals in APR and knowledge of AMR have also been reported by various other studies from other parts of the world , 56 and Our study data show that physicians practicing in paediatric department prescribed antibiotics more frequently than those from other departments, and physicians practicing in internal medicine and paediatric were more likely to prescribe the Reserve antibiotic Cefepime than those in other departments. Several PPS suggest that paediatric departments rank second only to the intensive care unit with respect to antibiotic use in hospital settings –41, 54. Our study had several limitations. First, we did not evaluate all aspects of AMS in healthcare facilities as defined by the WHO . HoweverIn conclusion, our study results suggest a lack of implementation of AMS among tertiary hospitals in Nigeria. Although most physicians working in these hospitals had above average KAP of APR and AMR, there were gaps in knowledge and attitude, as well as frequent and inappropriate prescriptions of antibiotics, including those in the Reserve group. Good KAPPr were associated with institutional, geographical, educational, and professional factors. Our study findings should inform interventions that optimize the use of antibiotics in healthcare facilities and improve awareness and understanding of AMR in Nigeria in line with Nigeria’s NAPAR.


In response to the accepted risk of emerging antimicrobial resistance, many organizations and institutions have developed and delivered events and activities designed to raise awareness of the issue and to change the behaviour of the intended audience. However, few of these events for a general public audience are documented or able to be sourced by those who might wish to repeat, adapt or modify, particularly those events that are successful. ‘Insider knowledge’ appears to be the best search tool. Moreover, evaluation of the success or impact of the event is rarely published. It would be useful if there were a ‘hub’ where descriptions of such activities could be deposited, enabling the building of a significant resource with real academic value. In January 2019, the UK Government published its 20 year vision for antimicrobial resistance (AMR),This is not a systematic review of the literature on public engagement with AMR (in part because so few activities have been published). Rather, we had to rely on personal experience and knowledge of activities delivered by colleagues and their contacts. Our single criterion for inclusion in this review was that the activity was searchable. In order to widen our search, 25 UK-based academics, artists and organizations known to the authors for AMR-related activities were directly approached (by e-mail), asking for citable examples of their relevant work. Seventeen responded , of whom 11 provided examples that are cited in this paper. Additionally, indirect evidence of events was sought via social media. Our findings are presented in the Results sections. They are divided into two sections, the first focusing on direct messages to the public from health professionals and scientists. The second describes a variety of cross-disciplinary collaborations, primarily involving arts and sciences, where AMR is addressed as part of the output.https://www.sciencedirect.com/) using pre-defined search terms was carried out. Search terms were required to feature in the ‘title, abstract or author-specified keywords’ to be included. Search terms can be found in Table In order to gain insight into the number of publications relating to the public engagement of AMR, a document search using the ScienceDirect search tool , led by PHE, asks people to pledge towards a series of behaviour changes around antibiotic prescribing, use and stewardship, infection prevention and control, self-care and health-seeking. The website has been heavily used ,European Antibiotic Awareness Day (EAAD) was launched in November 2008. England and Scotland participated, using an existing antibiotic awareness campaign that had previously run in February 2008. An evaluation of the November campaign (using a large ‘before and after’ public survey) found that whilst 83% of participants were potentially exposed to the campaign, there was no positive effect detected in terms of understanding the relationship between antibiotics and viral infection or reported use of antibiotics.http://antibiotic-action.com/), which was launched in 2011 and was wholly funded by BSAC, producing a range of resources designed to inform different stakeholders, from politicians to members of the public. A well-publicized national campaign was launched in 2017, aimed at reducing patient expectation for antibiotics and supporting a reduction in prescriptionsAn early evaluation of the programme suggests that while 69% of pledges came from healthcare professionals, and the remaining 31% from members of the public,www.nesta.org.uk). In 2013, a public vote in the UK selected AMR as the global challenge for which the prize should be awarded  with a goal to develop a transformative point-of-care test that is useable anywhere in the world to ensure correct and appropriate prescription of antibiotics . The public vote was launched with a special episode of Horizon, a long-standing BBC science documentary series, and announced 1 month later live on the prime-time BBC magazine-style The One Show. Whilst this exposure and encouragement of public involvement is likely to have engaged large numbers, no literature could be found that evaluates the impact the choice of AMR as the winning topic has had on public understanding and/or perception of the problem.The Longitude Prize reflects the national priority given to AMR. It is an international prize fund (£10 million) led by the UK-based challenge innovation foundation Nesta (www.theconversation.com) is an independent online news service, where academics write topical articles in a journalistic manner, which can then be accessed and reproduced by more than 22 000 websites with potential reach of tens of millions of people.Many individuals or organizations interested in discussing AMR with the public have used digital/social media to good effect. For example, The Conversation (https://e-bug.eu). These e-Bug resources are recommended by NICE for all schools to teach about hygiene, infections and antibiotics.The e-Bug programme is led by PHE, in cooperation with a consortium of 28 international partner countries. e-Bug aims to educate children and young people about microbiology, hygiene and the spread and treatment of disease. In order to reduce the incidence of antibiotic resistance, e-Bug also aims to reinforce awareness of the benefits of prudent antibiotic use and the impact of inappropriate use.https://gamedrlimited.com/about/) is an organization aimed at developing digital education/training solutions in the healthcare-related sector, including games related to AMR such as the Bacteria Combat game, available on Android and iOS, developed in collaboration with the University of Glasgow. Evaluation of Bacteria Combat, which included a mixed-methods approach of both qualitative and quantitative analysis, demonstrated a significant proportion (95%) of school students  found the game interesting. Further pilot trials demonstrated greater potential for engagement with multiplayer variants of the game.The Game Doctor (https://microbiologysociety.org/education-outreach/antibiotics-unearthed.html), launched following the success of the Small World Initiative (SWI), a USA college-level engagement programme. The aim of the SWI was to bring authentic research to schoolchildren to discover potential new antibiotics via culturing of soil and has been considered a success by educators.,Antibiotics Unearthed was a national campaign operated by the UK-based Microbiology Society (https://www.lstmed.ac.uk/public-engagement/swab-send). The programme, started in 2014,https://www.facebook.com/swabandsend/) show a wide geographical distribution, with participants in Scotland, England and Wales. The programme also aims to use research projects to engage primary and secondary school students with the challenges of AMR.http://www.ox.ac.uk/news/2015-09-25-microbe-artwork-shows-limits-antibiotics).Another citizen science-type activity is Swab and Send, initiated by UK academic Adam Roberts (https://seriouslyresistant.com). This has expanded to citywide community events, public media videos, outreach to schools, support from pharmacies across Leeds and national campaign launches. The Seriously campaign has over 18 000 pledges, as of August 2019.The Seriously campaign was launched in Leeds in 2016 to educate university students on the importance of correct use of antibiotics and linking with Antibiotic Guardians  and the Quadram Institute’s Guardians of the Gut (https://quadram.ac.uk/meet-the-guardians-of-the-gut) both allow walk-through immersive experiences.Museums and other venues such as town halls, arts centres and parks are often focal points for festivals, encouraging interdisciplinary events in order to attract different audiences. Many examples of AMR activities at festivals have already been noted above, but one recent example of immersive and cross-disciplinary outputs has been the gut microbiome and links of the human microbiome to AMR. The University of Salford’s MicrobiHome (https://wellcome.ac.uk/funding/schemes/public-engagement-fund). The Engineering and Physical Sciences Research Council (EPSRC) 2016 Bridging the Gaps AMR challenge  enabled multidisciplinary approaches to AMR, resulting in a range of innovative and engaging interdisciplinary activities as part of the work package outputs.There are also opportunities for members of the public to engage with AMR where non-scientific subjects provide an alternative viewpoint or output. A number of funding bodies also recognized the value of multi-disciplinary approaches to AMR and crossover ‘sci–art’ collaborations, notably Wellcome through its AMR programmes and public engagement funds (www.artscouncil.org.uk/what-we-do) for convenience: visual arts; collections; combined arts; dance; libraries; literature; museums; music; and theatre. The categories either address the creative discipline itself  or the vehicle through which creativity is demonstrated . Surprisingly or otherwise, we have identified activities designed to raise awareness of AMR in all of these categories, usually involving cross-disciplinary collaborations that might increase/provoke/inform the audience, and we have selected examples that illustrate the breadth of creative efforts focused on AMR. To simplify, we have combined categories and describe examples from the following: performance, visual arts, collections and literature.In this paper, we have categorized cross-disciplinary sci–art outputs separately, utilizing the categories identified by the Arts Council .Using a similar format, in 2017 a play entitled The Mould that Changed the World (http://mouldthatchangedtheworld.com/) is a musical produced in partnership between BSAC and the Charades Musical Company, in collaboration with the University of Edinburgh, and launched in 2018. It narrates the story of antibiotic discovery, the rise of antibiotic resistance and the importance of preserving antibiotics for now and the future. Local scientists take part in the production and schools are encouraged to develop their own musicals using a resource kit . A pre- and post-intervention survey was carried out in two schools and demonstrated that children had gained and retained knowledge 2 weeks following the musical.NosocomialArtibiotics  describes the battle between bacteria and antibiotics through music composed by Eduardo Miranda, Director of the Peninsula Arts Contemporary Music Festival, where the piece was performed in 2018. In the musical narrative, bacteria and antibiotics are presented as sound and the performance charts their quest to respectively damage and defend the DNA of their host. The project was commissioned by the research company Biofaction, and Miranda spent a period as artist-in-residence of the Synpeptide project, working alongside scientists at the Institute of Medical Microbiology and Hygiene in Regensburg, Germany. No information was available with regard to impact and evaluation.https://www.campaignlive.co.uk/article/public-health-englands-catchy-jingle-aims-danger-antibiotic-resistance-unforgettable/1447966).Continuing the musical theme, the PHE provided another musical angle with its jingle (https://gamedrlimited.com/) developed an innovative short film, Antibiotic Apocalypse, that uses dance and emotive storytelling. A collaborative venture with the University of Glasgow and the Liverpool School of Tropical Medicine, an Edinburgh-based choreographer was recruited to oversee dance production, developing the production alongside specific learning outcomes. Antibiotic Apocalypse was released on YouTube, Vimeo and social media platforms on Hand Hygiene Day 2017, was entered into several competitions and film festivals and screened across the UK, including in patient waiting rooms. The planning of the film, its release and evaluation were thorough and findings were informative.To engage young adults (aged 18–24 years) with AMR, Game Doctor  and Catch , similarly raise awareness of AMR. Resistance is a documentary outlining difficulties of controlling AMR, particularly due to issues with the use of antibiotics for livestock. The Society for Applied Microbiology (www.sfam.org.uk) sponsored a screening of the film with a panel of experts available to answer questions raised by the audience. Unfortunately, there is no documentary evidence of the event (which was well attended) and no evidence could be found for any impact of the film on audiences. Catch is more obviously designed to raise awareness of a post-antibiotic era and is eminently suitable for complementary discussion. The film was written and directed by Paul Cooke and Dominic Rees-Roberts, participants of the Wellcome Trust’s Emerging Talent programme for science communicators, and is backed by the Royal College of Pathologists and leading scientists from the University College London (UCL) Centre for Clinical Microbiology, the University of Cape Town, the University of Birmingham and Antibiotic Action.Two films, Resistance,The Truth about Hawaii,Michael Mosley versus the Superbugs (https://www.renegadepictures.co.uk/program/michael-mosley-vs-the-superbugs_1487.aspx), in which he worked with artist Mellissa Fisher to demonstrate how the skin microbiome could acquire resistance using a human-sized agar sculpture, ‘microbial Michael’ (www.mellissafisher.com). Similarly, Angela Rippon’s The Truth About AntibioticsThere have been several television and radio programmes focusing on AMR, either as documentary programmes or drama. BBC Radio 4 has recently broadcast two radio series: In short, there is a relatively large and well-documented fund of performance and media outputs in existence that could be used to encourage engagement with AMR amongst a range of audiences, yet there is perhaps a lack of information on facilitative learning materials that might enable professionals to use them effectively.https://labiotech.eu/bioart/anna-dumitriu-crispr-antibiotic-resistance/ and https://labiotech.eu/bioart/bioart-london-anna-dumitriu/). In one project, she worked with a team of scientists to help create a method to safely display a collection of ‘superbugs’ commissioned for the Science Museum London’s major exhibition ‘Superbugs: the Fight for Our Lives’. The exhibit was on show between November 2017 and April 2019 and was viewed by over 1.5 million visitors. A reviewSAW AntibioticsThere are a number of artists who use microbiology within their practice and several of them have focused on AMR, either independently or as an artist-in-residence. For example, Anna Dumitriu (annadumitriu.tumblr.com) has completed an impressive number of works, many of which touch upon AMR , the audience (jury) were asked to vote on the motion. Interestingly, the number of respondents who believed in a solution fell after the event, perhaps reflecting that a more informed—and less optimistic—opinion had been made. Jury engagement related to the talent of the expert witnesses rather than to the content.,https://www2.mmu.ac.uk/engage/what-we-do/bad-bugs-bookclub/) comprises scientists and non-scientists who read novels in which infectious disease forms part of the plot. Reading guides and meeting reports are provided for all of the 60 books so far discussed by the group. There are relatively few novels that feature AMR, perhaps because the infections tend to affect individuals rather than populations. Some rely on a terrorism-type activity where an antibiotic-resistant bacterium is deliberately introduced into a clinical setting .Surgeon X,Infectious FuturesA Fierce RadianceThere have been several popular science books published about AMR ,www.ifh-homehygiene.org) has recently published its own policy document (http://www.ifh-homehygiene.org/review-best-practice/hygiene-hypothesis-and-its-implications-home-hygiene-lifestyle-and-public), where they emphasize the importance of, and differentiation between, hygiene and cleanliness.,Catch, the movie Resistance, the Infectious Futures short stories and the radio series The Trouble With Hawaii all enable encounters with individuals affected by AMR.It is widely recognized that AMR is an issue that will critically affect future health and there are many schemes, programmes, initiatives and events taking place that are designed to raise awareness amongst the intended audience and change their behaviour. Implicit in all of these activities should be the identification of the key hypothesis/message and the particular audience, the design and delivery of the activity, and an evaluation of its success. Initiatives like the Antibiotic Guardian campaign have identified specific messages that enable activities to be tailored to fulfil aims. Interestingly, the International Scientific Forum on Home Hygiene  and second (‘improve hygiene and prevent the spread of infection’) commandments of the O’Neill report,The larger national initiatives were developed to address key messages for their audiences and they do include significant evaluations. They also provide resources to support those who are delivering events as well as good models for those who wish to develop their own resources and activities. It is also relatively easy to obtain/analyse data that demonstrate effectiveness of initiatives delivered through digital and social media. Markers of success do reveal knowledge and behavioural changes that contribute to the overall ‘fight against AMR’. It is important that we, as ‘experts’, deliver effective activities and ensure that they are shared with others and that they are critically evaluated to ensure maximum effectiveness and impact. It has been noted that there is an urgent need for microbial literacy in societyNone to declare.dlaa012_Supplementary_DataClick here for additional data file.


The role of wildlife with long-range dispersal such as gulls in the global dissemination of antimicrobial resistance (AMR) across natural and anthropogenic aquatic environments remains poorly understood. Antibiotic-resistant bacteria have been detected in resident and migratory gulls worldwide for more than a decade, suggesting gulls as either sentinels of AMR pollution from anthropogenic sources or independent reservoirs that could maintain and disperse AMR across aquatic environments. However, confirming either of these roles remains challenging and incomplete. In this review, we present current knowledge on the geographic regions where AMR has been detected in gulls, the molecular characterization of resistance genes, and the evidence supporting the capacity of gulls to disperse AMR across regions or countries. We identify several limitations of current research to assess the role of gulls in the spread of AMR including most studies not identifying the source of AMR, few studies comparing bacteria isolated in gulls with other wild or domestic species, and almost no study performing longitudinal sampling over a large period of time to assess the maintenance and dispersion of AMR by gulls within and across regions. We suggest future research required to confirm the role of gulls in the global dispersion of AMR including the standardization of sampling protocols, longitudinal sampling using advanced satellite tracking, and whole-genome sequencing typing. Finally, we discuss the public health implications of the spread of AMR by gulls and potential solutions to limit its spread in aquatic environments. Antimicrobial resistance (AMR) is a major global health challenge affecting human, animal, and environmental health . Thus, aLeucophaeus pipixcan), migrating across America from Canada to Chile, could result in the dissemination of ARB and ARGs over extensive geographic areas, dispersing AMR from regions with high levels of AMR to less affected areas  receivers of ARB or antibiotic-resistant genes (ARGs) and acting as sentinels of human environmental pollution to natural ecosystems  or as  checklist . All aut∗ OR antimicrobial resist∗), , and (bacteria). Each of them was merged with (marine bird∗ OR aquatic bird∗ OR gull∗ OR Larus). Details of the search strategy are available as an additional file  showing the presence and/or potential transmission of ARB and ARGs. Thus, we included only studies providing at least one of the following information: (i) wild gull species where ARB was recovered, (ii) phenotypic resistance to specific antibiotics in bacteria isolated from gulls, and/or (iii) ARGs identified in bacteria isolated from gulls. There were no restrictions related to the year of publication or geographical location. Any type of reviews or studies including After the removal of duplicates, we identified a total of 3,475 articles published from 1964 to January 2021, including 3 additional references that were identified from reading these papers. Pre-selection by title and abstract reduced to 227 articles for full-text analysis, and 90 fulfilled the preestablished criteria and were included in the final analysis . The remExtracted data were independently performed by three authors  and verified by other authors. Disagreements were resolved through discussion. The obtained data were entered into a Microsoft Excel template adapted from a previous study . This fiWe estimated the proportion of studies filling a given criteria  using R. 3.1.6 (R Development Core Team).Our scoping review identified 90 articles published between 1981 and 2020, although only 22% of these studies were published before 2010. The number of studies published on gulls increased from 1 in 1981 to 10 per year in 2020 and peaked in 2017 with 12 articles . StudiesLarus argentatus, 26%), followed by the laughing gull  and the yellow-legged gull   . These tEscherichia coli (59%), Salmonella enterica (23%), Campylobacter jejuni (8%), and Klebsiella pneumoniae (8%)  . The temLarus dominicanus (100%), while Proteus mirabilis showed the highest proportion of positive individuals (27.9%)  was estimated in one study in Africa (70%) that included less than 50 individuals. The highest proportion of positive gulls was observed among  (27.9%) . Given t (27.9%) . Overall (27.9%) , includi (27.9%) . In partCampylobacter spp. (6%) and Salmonella spp. (8%) both resistant to fluoroquinolones were the most identified. No “medium-priority” pathogen has been recovered from gulls.Among the four antimicrobial-resistant pathogens considered as a “critical priority” by the WHO , all werARGs were reported in 70% of studies conducted in gulls , 4. MobiblaCTX–M. Studies detected blaCTX–M–14 and blaCTX–M–15 in Asia, Europe, North and South America, and Oceania. Likewise, blaCTX–M–55 was reported in all these continents with the exception of South America. Beta-lactamases blaCMY–2 (AmpC) were reported in Europe, North America, and Oceania, and blaOXA–48 (carbapenemase) were reported in Africa, Europe, North America, and Oceania (Most studies detecting ARGs focused on beta-lactamase genes including extended-spectrum beta-lactamases (ESBL), AmpC-type beta-lactamases, and carbapenemases, which were identified in all continents but Antarctica . Among t Oceania . Of the  Oceania . Asia onOnly 19% of studies suggested a potential origin for the ARB or ARGs detected among gulls. Landfill (41%), places close to gulls nesting, and/or resting areas with high human density (29%), sewage effluents (29%), and contaminated water (6%) were suspected. Suspicions were based on potential contamination sources around the sampling area. However, only one study  carried E. coli isolates between different locations. Their results showed that the prevalence and genetic typing of AMR isolates were highly similar between gulls and a landfill. Salmonella since all positive individuals were negative during the sampling 2 months later.Two studies (2%) tagged gulls in both the origin and final movement areas to identify whether they were capable of spreading AMR across the landscape . AhlstroL. argentatus and C. ridibundus) and most on E. coli and Salmonella spp. Despite ARB and ARGs being widely detected in gulls, our analyses showed that the origin of these AMR remains unknown in 81% of studies, and only two studies followed gulls across time (for up to 3 years), but none has been able to prove that gulls were reservoirs  to assess potential cross-species transmission of ESBL-E. coli , Staphylococcus aureus vancomycin-intermediate or -resistant (high priority), and Shigella spp. fluoroquinolone-resistant (medium priority) could reflect a lack of research and not necessarily that these bacteria are not circulating among gulls. In fact, one study using a metagenomic approach found 31 previously undescribed ARGs, while another detected more than 70 bacterial species and 24 ARGs . Thus, the absence of other global-priority ARB in current studies such as  24 ARGs . This re 24 ARGs . The pre 24 ARGs . Our revE. coli ST131, previously associated with nosocomial infections in humans, has been identified in gulls mainly from the United States . Secondly, future research should identify if environmental factors such as plastic and heavy metal pollution are also selecting ARB and ARGs in gulls independently of contact with humans. Thirdly, the clinical relevance and conservation implications of the detected ARB for gull’s health require further investigation. In particular, there is no evidence that the observed bacteria cause any pathogenicity to the studied gulls nor complicate treatment of gulls with antibiotics in rehabilitation centers. Finally, innovative techniques such as satellite tracking and collaborations across research teams in different countries where gulls migrate  could help elucidate whether gulls are spreading ARB and/or ARGs across countries and continents during their migration.JB and DZ-G: conceptualization. DZ-G: data curation. JB, DZ-G, ZR-S, and MS-C: formal analysis, investigation, and methodology. JB: funding acquisition, project administration, resources, software, supervision, validation, and visualization. DZ-G, JB, ZR-S, MS-C, CT, and PP: writing—original draft and writing—review and editing. All authors have read and agreed to the published version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Drug addiction is defined as a compulsive pattern of drug-seeking- and taking- behavior, with recurrent episodes of abstinence and relapse, and a loss of control despite negative consequences. Addictive drugs promote reinforcement by increasing dopamine in the mesocorticolimbic system, which alters excitatory glutamate transmission within the reward circuitry, thereby hijacking reward processing. Within the reward circuitry, the striatum is a key target structure of drugs of abuse since it is at the crossroad of converging glutamate inputs from limbic, thalamic and cortical regions, encoding components of drug-associated stimuli and environment, and dopamine that mediates reward prediction error and incentive values. These signals are integrated by medium-sized spiny neurons (MSN), which receive glutamate and dopamine axons converging onto their dendritic spines. MSN primarily form two mostly distinct populations based on the expression of either DA-D1 (D1R) or DA-D2 (D2R) receptors. While a classical view is that the two MSN populations act in parallel, playing antagonistic functional roles, the picture seems much more complex. Herein, we review recent studies, based on the use of cell-type-specific manipulations, demonstrating that dopamine differentially modulates dendritic spine density and synapse formation, as well as glutamate transmission, at specific inputs projecting onto D1R-MSN and D2R-MSN to shape persistent pathological behavioral in response to drugs of abuse. We also discuss the identification of distinct molecular events underlying the detrimental interplay between dopamine and glutamate signaling in D1R-MSN and D2R-MSN and highlight the relevance of such cell-type-specific molecular studies for the development of innovative strategies with potential therapeutic value for addiction. Because drug addiction is highly prevalent in patients with other psychiatric disorders when compared to the general population, we last discuss the hypothesis that shared cellular and molecular adaptations within common circuits could explain the co-occurrence of addiction and depression. We will therefore conclude this review by examining how the nucleus accumbens (NAc) could constitute a key interface between addiction and depression. Drug addiction is defined as a compulsive pattern of drug-seeking/taking behavior despite damaging consequences, as well as a high rate of relapse after an abstinence period. Over the past years, research on preclinical models of addiction established that pathological behavior induced by addictive substances originate from long-lasting alterations of synaptic transmission . Drug-evA hallmark of drugs of abuse is to artificially increase dopamine concentration in the mesocorticolimbic system , which ps/olf and Gi/o subtypes  and downstream protein kinase A (PKA) pathway in D1R-MSN, while repressing it in D2R-MSN. When dopamine concentration rises, multiple PKA targets, including glutamate receptors, are therefore regulated in opposite ways in both MSN populations, which leads to a facilitation of glutamate-dependent activation of D1R-MSN and to its inhibition in D2R-MSN  imaging performed in the dorsal striatum of anesthetized drd1a-eGFP or drd2-eGFP mice showed that an acute cocaine injection produces a fast increase of intracellular Ca2+ in D1R-MSN, while a progressive decrease is observed in D2R-MSN  are Ca2+ permeable ionotropic glutamate receptors that have long been recognized as essential for synaptic plasticity, as well as learning and memory  pathways in the striatum in response to acute cocaine exposure , which reinforces the phosphorylation of MEK and ERK . Although SUD are complex multifactorial psychiatric conditions, stressful experiences constitute a primary factor that can potentiate responses to most licit and illicit abused substances and therefore favor the appearance of addictive behaviors . Importavia activation of glucocorticoid receptors.Epidemiological and clinical data indicate that people suffering from SUD often experience, at least, a second psychiatric conditions, distinct from drug abuse, such as mood disorders. When two pathologies occur, simultaneously or sequentially, in the same person, they are described as comorbid. In addition to time correlation, comorbidity also implies interactions between the illnesses that affect the course and prognosis of both. A major finding in psychopathology research is that comorbidity is the rule rather than the exception. Comorbid SUD and psychiatric disorders are indeed commonly encountered in our modern society. However, they often lack adequate treatment and are mostly associated with poor prognosis. Therefore, comorbidity has emerged as a major clinical, public, and research issue . The higSeveral models of stress with various degrees of duration and intensity have been developed in order to mimic symptoms reminiscent of human mood disorders including depression . AlthougAs largely evidenced in the sections related to drugs of abuse, dopamine has a key role in filtering excitatory inputs to the NAc. Similarly, stress has been shown to dysregulate excitatory inputs to the NAc in a projection-specific manner. Indeed, an enhancement of thalamic-NAc and ventral hippocampal-NAc synaptic transmission was observed in mice susceptible to CSD . Conversvia its action on the melanocortin type 4 receptor (via Sh-RNA was sufficient to reverse stress-induced anhedonia (in vivo activity of D1R-MSN; indeed, increased excitability of D1R-MSN was reported following CSD. Activation of D1R-MSN via optogenetic stimulation reversed social aversion, while their pharmacogenetic silencing conferred stress susceptibility (Examining stress-induced cell-type-specific adaptations in the NAc, chronic restraint stress was shown to decrease AMPA/NMDA ratio in D1R-expressing neurons, while D2.R-expressing neurons remained unaffected . The autreceptor . Targetinhedonia . Within nhedonia . Furthertibility . In conttibility . These dtibility . Increastibility . Strikintibility . RegardlScarce studies examined the impact of stress on inhibitory synapses within the NAc. Recently, a decreased expression of neuroligin-2 was reported in post-mortem tissue analyses from the NAc of depressed patients . These fAlterations in MSN cellular architecture has been associated with dopamine-dependent neuronal adaptations , especiaSIRT1 locus, which encodes a histone deacetylase (fosb gene with distinct contribution of acetylation and methylation in D1R-MSN and D12R-MSN (The differential impact of stress on D1R-MSN and D2R-MSN are also found at the molecular and epigenetic levels. Indeed, alterations in gene expression programs underpin enduring physiological and behavioral adaptations that contribute to the appearance of depression symptomatology. Recently, a whole-genome sequencing of patients with recurrent major deficit disorders identified a significant increase of the signal for the cetylase . Increascetylase . Of intecetylase . ΔFosB ecetylase , but it cetylase  and its cetylase . In contcetylase . This baD12R-MSN .Overall, this review highlights the complexities of adaptations induced by natural and addictive rewards triggered within the NAc emphasizing the differential contribution of D1R-MSN and D2 MSNs. We described how stress also highjacks the reward system to induce adaptations at specific inputs to the NAc and also in a cell-type-specific manner. Although, much work is needed to build a comprehensive landscape of the enduring changes occurring following these stimuli, the development of viral tools in order to probe for cell- and projection-specific adaptations has already allowed to decipher some of the key sequences of events that underlie pathological states based on the deregulation of the reward system. Facing the high prevalence of comorbidities between SUD and other psychiatric disorders, with about 50% of patients suffering from one condition also developing the other, it has been proposed that brain dysfunctions underlying SUD and other mood disorders, such as depression, may share partly common pathophysiological mechanisms. Future work should therefore focus on the identification of common molecular events that are essential for SUD and other psychiatric conditions. This is in line with the recent push toward transdiagnostic approach of psychiatric disorders and recommendations, notably from the NIDA, stressing that therapeutic developments should focus on both SUD and comorbid mood disorders, rather than treating each disease independently, which holds poor efficacy .All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The deletion breakpoints (determined with whole-genome linked-read sequencing) were in the FKTN gene  and in a non-coding area  (hg19). The deletion encompassed six protein-coding genes . ZNF462 haploinsufficiency was consistent with the patient’s Weiss–Kruszka syndrome , but did not explain his KS. In further analyses, he did not carry rare sequence variants in 32 known KS genes in whole-exome sequencing and displayed no aberrant splicing of 15 KS genes that were expressed in peripheral blood leukocyte transcriptome. The deletion was 1.8 Mb upstream of a KS candidate gene locus (PALM2AKAP2) but did not suppress its expression. In conclusion, this is the first report of a patient with Weiss–Kruszka syndrome and KS. We suggest that patients carrying a microdeletion in 9q31.2 should be evaluated for the presence of KS and KS-related features.Patients with deletions on chromosome 9q31.2 may exhibit delayed puberty, craniofacial phenotype including cleft lip/palate, and olfactory bulb hypoplasia. We report a patient with congenital HH with anosmia  and a  Congenital hypogonadotropic hypogonadism (CHH) is a rare genetic disease that prevents pubertal development and elicits infertility due to deficient secretion or action of gonadotropin-releasing hormone (GnRH) . CHH is ZNF462 mutations or microdeletions in the 9q31.2 area  had been assessed for delayed development and subsequently for the absence of puberty. He was the second child of healthy nonconsanguineous parents , and he ANOS1, CHD7, FGF8, FGFR1, GNRHR, KISS1R, PROK2, PROKR2, and TACR3 up to20 bp of the intronic sequence with single nucleotide changes, small indels up to 220 bp, and copy number variations defined as single exon or larger deletions and duplications.At the age of 13.5 years, the proband was referred for the evaluation of delayed puberty and was noted to have a hypoplastic scrotum and testes. He had self-reported anosmia, which had been well appreciated in the family and absent olfactory bulbs in the MRI. He had no synkinesia, hand or foot deformities, pigmentation defects or missing teeth, nor he reported problems with balance. Abdominal ultrasound revealed two kidneys. His circulating reproductive hormone levels were low . At the age of 14 years and 11 months, he was still prepubertal (Tanner stage G1) with pubic hair stage P3. His testes were very small, only 1 cm in length (corresponding to < 1 mL). His reproductive hormone levels were still low; serum LH 0.1 IU/L, inhibin B 23 ng/L, and testosterone 0.4 nM. Based on the very small testicular size, low reproductive hormone and inhibin B levels, anosmia, and the absence of olfactory bulbs in the MRI scan, he was prepubertal, and a diagnosis of Kallmann syndrome was set . InductiANOS1, FGFR1, FGF8, FGF17, PROK2, PROKR2, CHD7, NSMF, HS6ST1, WDR11, SEMA3A, SEMA7A, PLXNA1, SOX10, IL17RD, FEZF1, NDNF, TCF12, TUBB3, DCC, SMCHD1, KLB, NTN1, SPRY4, PTCH1, FLRT3, AMH, DUSP6, PLXNA3, NRP1, SPRY2, and NRP2) with Loupe 2.1.1.DNA of all family members was extracted from peripheral blood leukocytes (PBL). The proband and parent RNA were isolated with the QIAamp RNA Blood Mini Kit (QIAGEN) from PBLs. The whole-genome linked-read sequencing of the proband was performed at FIMM (Institute for Molecular Medicine Finland) according to 10× Genomics Chromium library preparation . The sample was sequenced with NovaSeq 6000 system  using S4 flow cell and XP workflow. Read length for the paired-end run was 2×151. The reads were aligned to the GRCh37 (hg19) reference genome. The data were analyzed with Longranger 2.2.2 WGS pipeline with default parameters and GATK for variant calling and visualized with Loupe 2.1.1 (both programs by 10× Genomics). The genome was covered with a mean depth of 34.7×, and a mean molecule length of 26.8 kb. Loupe interactive visualization tool 2.1.1 was employed to define the exact 9q31.2 deletion breakpoints. In addition, as MLPA (multiplex ligation-dependent probe amplification) is available for only a fraction of known KS genes, we verified the absence of genomic structural variants of over 40 bp in size in the coding regions of 32 known KS genes  according to the manufacturer’s instructions in a regular thermocycler with 1 µg of total RNA. The synthesized cDNAs were used as templates to assess the mRNA expression with a ready-to-use qPCR mix (Solis Biodyne) in a quantitative PCR machine (Roche LC480 II). The expression levels of ANOS1, FGFR1, FGF8, PROK2, PROKR2, CHD7, and WDR11  and AKAP2  were amplified with PCR from the genomic DNA. The PCR conditions and primers are available upon request. The PCR products were purified with ExoProStar treatment , and sequenced from both directions using the ABI BigDyeTerminator Cycle Sequencing Kit (v3.1) and ABI Prism 3730xl DNA Analyzer automated sequencer (Applied Biosystem). The DNA sequences were aligned and read with Sequencher® 4.9 software . As one of the patients harbored a variant NC_000009.11:g.112918586C>T, in intron 2 of AKAP2, his RNA was converted into cDNA by using the SuperScript® III First-Strand Synthesis System for RT-PCR kit (Invitrogen by Life Technologies) according to the manufacturer’s instructions. Part of the AKAP2 transcript was PCR-amplified with cDNA-specific primers and subsequently sequenced. Effect of the AKAP2 variant NC_000009.11:g.112918586C>T was also predicted with Human Splicing Finder (https://www.genomnis.com/access-hsf) (http://www.mutationtaster.org/) (http://www.cbs.dtu.dk/services/NetGene2/) (https://www.fruitfly.org/seq_tools/splice.html) (http://wintervar.wglab.org/)  without mutations in nd WDR11 , 20. Theess-hsf) , Mutatioer.org/) , NetGenetGene2/) , 24, andce.html) , and itsab.org/) .The whole-exome sequencing (WES) of the proband’s family was performed with Illumina Novaseq S2 PE100 technology. First, the adapter was trimmed from the reads, as well as any low-quality nucleotides from the 5’ or 3’ ends of the read, removing pairs with less than 36 bp. The reads were aligned to the GRCh37 (hg19) reference genome with the BWA (Burrows–Wheeler Aligner). Non-unique read pairs and non-unique single reads were removed and GATK Base Recalibrator was used to clean the alignment. Any potential PCR duplicates were removed using Picard MarkDuplicates, and GATK IndelRealigner was used for indel sites. The mpileup from the SAMTOOLS package was used for variant calling . The seqANOS1, FGFR1, FGF8, FGF17, PROK2, PROKR2, CHD7, NSMF, HS6ST1, WDR11, SEMA3A, SEMA7A, PLXNA1, SOX10, IL17RD, FEZF1, NDNF, TCF12, TUBB3, DCC, SMCHD1, KLB, NTN1, SPRY4, PTCH1, FLRT3, AMH, DUSP6, PLXNA3, NRP1, SPRY2, and NRP2) were covered in WES by using the BasePlayer analysis and visualization tool (https://doc-openbio.readthedocs.io/projects/annovar/en/latest/). We defined that a potentially causative variant should be in the consensus splice-site or it should be nonsynonymous and not classified as benign by any of the ten applied in silico tools . Additionally, a potentially causative variant(s) should (i) occur only in the proband (de novo) ; (ii) be biallelic  (MAF <2%); (iii) be monoallelic and inherited from one parent and be absent in the sister  (MAF <0.1%); or (iv) be monoallelic and inherited in the X chromosome (X-linked recessive) (MAF <2%). The frequency criteria were applied for all ethnic subpopulation frequencies in gnomAD, ExAC, 1000 Genomes, and Exome Variant Server (provided in the ANNOVAR annotation) and in dbSNP (https://www.ncbi.nlm.nih.gov/snp/)  and SARS1  (GRCh37). The PCR conditions and primers are available upon request.First, we verified that the coding exons of 32 KS-associated genes   . Following the quality control, RNASeq libraries were prepared using NEBNext Ultra II Directional Poly (A) Capture Kit. RNA sequencing was carried out as paired-end sequencing with Illumina NextSeq 500 Mid Output System. RNA quality-testing, library preparation, and sequencing were performed in the Functional Genomics Unit (FUGU) at the University of Helsinki, Finland.Raw adaptor-trimmed FASTQ files were subjected to quality assessment with the FASTQC tool . Based on the overall data quality and the individual reads, additional trimming was unnecessary. The sequences were aligned to the GRCh37 human genome version with the STAR package . SplicinPALM2AKAP2 in the PBLs of the proband (n = 4) and his parents (n = 4) were compared with one-way ANOVA, and followed by Tukey’s HSD post-hoc analysis. P-value < 0.05 was accepted to indicate statistical significance. To evaluate the role of the 9q31.2 microdeletion in our proband, we utilized the CNV data obtained from the UK Biobank  , none of which has been implicated in KS.To define the exact deletion breakpoints, we performed a whole-genome linked-read sequencing of the proband’s DNA. He carried a 2.38 Mb deletion on chromosome 9:108,331,353–110,707,332, excluding 2 (hg19) . InsteadPALM2AKAP2 has been implicated in KS and our proband’s deletion laid approximately 1.8 Mb upstream of the PALM2AKAP2 locus , with a frequency of 0.00004267 in Finns  (http://wintervar.wglab.org/)) , we confirmed in vitro that the splicing was normal (data not shown). All other encountered PALM2 and AKAP2 variants had MAFs above 1% among gnomAD Finns.To further probe the possible role of ng genes , 20. In te.org/) . This vaper se, or the adjacent candidate gene PALM2AKAP2 provided no equivocal answer to our question of whether this deletion underlies KS in the proband, we sequenced the whole exome in the proband, his healthy parents and sister. First, no rare sequence variants were found in the 32 genes implicated in KS . Secondly, we investigated in the linked-read WGS data whether the proband carried genomic structural variants of over 40 bp in the coding regions of these 32 KS genes. However, none of the genes were disrupted by structural variants. As WES may not detect non-coding variants that affect splicing, we investigated the splicing events of KS genes in the proband and his parents with RNA sequencing and subsequent visualization of transcripts. Overall, 15 KS genes, FGFR1, PROK2, CHD7, NSMF, HS6ST1, WDR11, SEMA3A, SEMA7A, PLXNA1, TCF12, SMCHD1, PTCH1, and DUSP6, were expressed in PBLs, and none of them displayed aberrant splicing events (data not shown). Finally, we investigated whether the WES data would contain new potential KS candidate gene mutations. Two rare variants of uncertain significance emerged, one in RIMBP3C and the other in SARS1. An overview of the variants is shown in Supplementary Table 1 and Given that the targeted KS gene panel, the deletion in 9q31.2 RNA5SP293. According to the UCSC Genome Browser (https://genome.ucsc.edu/), the following transcription factors were determined to have binding sites in this overlapping region: GATA3, MYC, JUND, CEBPB, TEAD4, GATA2, MAFF, USF2, USF1, BHLHE40, ZNF143, ATF3, and SIX5.Our proband’s deletion  and the deletion of the previously described patient with CHH  . The deletion encompassed loci of six protein-coding genes .We report a patient with KS, developmental delay, ptosis, ventral septal defect, craniofacial phenotype, and mild bilateral sensorineural hearing loss without coding region mutations in the established KS genes, which was assessed by using a targeted sequencing panel, linked-read whole-genome sequencing, RNA sequencing, and WES. Instead, he carried a heterozygous FKTN in the first intron. However, to the best of our knowledge, FKTN is not implicated in KS or CHH , the nearest genes to this SNP being TMEM38B, FKTN, TAL2, and ZNF462  is relatively far away, 1.8 Mb, downstream from the 3’ breakpoint of the deletion. This locus in our patient with KS was, however, spared and expressed PALM2AKAP2 efficiently , JUND (-1 cells  and is e-1 cells ), GATA2 -1 cells ) and CEBng cells  and is k in mice ). Howeveiciently . Moreovede novo deletion in 9q31.2 and KS. ZNF462 haploinsufficiency was consistent with the patient’s Weiss–Kruszka syndrome but did not explain his KS. In further analyses, he did not carry rare sequence variants in known KS genes. Our results suggest that patients carrying a microdeletion in 9q31.2 should be evaluated for the presence of KS and KS-related features, and conversely, that KS patients with features of Weiss–Kruszka syndrome should be evaluated for the presence of a heterozygous 9q31.2 microdeletion.Taken together, variation in the chromosomal region 9q31.2 at the population level associates with the variation in puberty timing and the sense of smell. Previous rare patients with deletions including 9q31.2 have exhibited CHH and KS-related phenotypes such as delayed puberty, an olfactory bulb defect, and cleft lip and/or palate. We describe the first patient with an extremely rare The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this study.Academy of Finlandhttp://dx.doi.org/10.13039/501100002341, Foundation for Pediatric Research, Sigrid-Juselius Foundation, Novo Nordiskhttp://dx.doi.org/10.13039/501100004191 Foundation, Emil Aaltonen Foundation, University of Helsinki, Helsinki University Hospital, and Päivikki and Sakari Sohlberg foundation. Taneli Raivio is on the Editorial Board of European Journal of Endocrinology.This work was supported by the Informed consents were obtained from all patients, and in the case of a minor/children also a parent or guardian gave the consent. The study was approved by the Ethics Committee of the Hospital District of Helsinki and Uusimaa and was conducted in accordance with the Declaration of Helsinki.PALM2AKAP2 RT-qPCR analyses; A-I P, J K, V Y, K V and T R wrote the manuscript, and all authors critically revised the manuscript. T R coordinated and managed the study.T R, V S, K V collected the study subjects and the clinical data and phenotyped the patients. J K, A-I P, V Y, H A, K V and T R analyzed the next-generation sequencing data, H A carried out ANNOVAR annotation, A-I P and K V performed Sanger sequencing data analyses. V Y performed


Systems neuroscience is focused on how ensemble properties in the brain, such as the activity of neuronal circuits, gives rise to internal brain states and behavior. Many of the studies in this field have traditionally involved electrophysiological recordings and computational approaches that attempt to decode how the brain transforms inputs into functional outputs. More recently, systems neuroscience has received an infusion of approaches and techniques that allow the manipulation  and imaging  of neurons, neurocircuits, their inputs and outputs. Here, we will review novel approaches that allow the manipulation and imaging of specific molecular mechanisms in specific cells (not just neurons), cell ensembles and brain regions. These molecular approaches, with the specificity and temporal resolution appropriate for systems studies, promise to infuse the field with novel ideas, emphases and directions, and are motivating the emergence of a molecularly oriented systems neuroscience, a new discipline that studies how the spatial and temporal patterns of molecular systems modulate circuits and brain networks, and consequently shape the properties of brain states and behavior. Systems neuroscience studies are focused on how particular circuits and large brain networks modulate brain states and behavior. Evolution has shaped circuits and brain networks to include a variety of cell types and a wide range of complex molecular properties. For example, within each cell of these networks, hundreds of thousands of molecules form complex systems that modulate the cellular responses that shape the output of these networks. Novel powerful techniques, that allow the tracking and manipulation of these molecular systems, in a cell specific manner and in real time, have fueled the emergence of molecular systems neuroscience, a sub-discipline of systems neuroscience that studies how the spatial and temporal patterns of molecular systems modulate circuits and brain networks, and consequently shape the properties of brain states and behavior. Molecular systems neuroscience studies attempt to explain brain processes by connecting molecular mechanisms to cellular and systems mechanisms in multiple brain regions engaged in processing information and generating behavior. Here, we will introduce this new field and review key examples of the exciting technological developments that motivated its emergence.Systems neuroscience is traditionally focused on how the activity of neuronal circuits gives rise to internal brain states and behavior. Many of the studies in this field have typically involved recordings of action potentials and other electrophysiological properties in specific brain regions, such as the visual cortex and hippocampus of awake behaving animals. The principal goal of these studies has been to explain how a particular electrophysiological phenomenon accounted for the observed behavior. For example, the discovery in the hippocampus of neurons that fire in specific places  was compelling evidence for cognitive maps , 2. In aMore recently, systems neuroscience has received an infusion of approaches and techniques that allow the manipulation  the processes that assemble circuits during development, and (b) the mechanisms that modulate their function in adult organisms. Inspired by evolution, insights on the function of genes in specific cells, cell systems and brain regions, may allow us to cut through the ever-growing complexity of how systems affect internal states and behavior.Second, besides electrophysiological properties, other cell biological properties of neurons and other cells, such as mechanisms of transcription, translation, neuromodulation, receptor function, etc. also have a critical modulatory role in behavior. Indeed, there is growing evidence for the reasonable hypothesis that the orchestration of these mechanisms in neuronal ensembles is critical for internal brain states and behavior. Therefore, imaging and manipulating these molecular mechanisms has already and will continue to yield insights into how cellular systems control behavior.Finally, there is now compelling evidence that the complex properties of behavior can be traced back to mechanisms that involve nearly every cell type in the brain, including, for example, astrocytes, oligodendrocytes and microglia. It is also reasonable to propose that the careful temporal and spatial organization of the myriad of molecular mechanisms regulating and orchestrating the function of these different cell types in the brain could make unique contributions to internal states and behavior that go well beyond the important contributions of neurocircuits. Accordingly, emerging molecular manipulation and imaging technologies, that together endow unprecedented cellular, spatial and temporal specificity, are being used to study the behavioral contributions of a number of molecular mechanisms in brain cell types other than neurons. The key thesis for a more molecularly oriented and broader systems neuroscience is that evolutionarily shaped molecular mechanisms in these cell systems may help explain and predict the vexing complexity of the brain’s internal states and behavior.New technologies have traditionally been at the heart of the generation and evolution of new perspectives and transformative discoveries in science. Next, we will review an exciting and powerful new set of molecular imaging and manipulation techniques that are transforming multiple fields in biology, and that we propose are fueling the emergence of molecular systems neuroscience. These new techniques allow for the unprecedented temporal, cellular and subcellular manipulation and imaging of specific molecular mechanisms in targeted cell systems, circuits and brain regions of behaving animals.New tools, methods and approaches unfailingly change a field by providing novel perspectives and opportunities for study. We identify three major areas of novel technology development that we predict will have a significant impact in systems neuroscience: (a) new developments in in vivo imaging techniques, including imaging of molecular mechanisms in specific cell types besides neurons, (b) new molecular sensors that can report on the activity of specific molecular mechanisms in a cell specific manner, and (c) new molecular actuators that can either activate or inhibit molecular mechanisms at a cellular or even sub-cellular level. These dramatic recent developments are opening systems neuroscience to more molecular and less neuron-centric perspectives.From an imaging standpoint, one of the significant innovations of the last decade was the introduction of miniaturized head mounted fluorescent microscopes (miniscopes) that can record from hundreds of neurons (or other cells) for many days in freely moving and behaving animals, including mice –24. ThisImaging with miniscopes involves removal of a small segment of the skull and possibly also brain tissue overlaying the imaging target, as well as the use of an implanted lens, such as a relay lens, to facilitate visualization of the target cells by the CMOS camera. Amongst other things, imaging with miniscopes facilitates the simultaneous visualization of a large population of cells, allowing for the detection of coordinated activity among groups of cells and thus identifying networks of communication within a cell population. Importantly, the nature of this type of imaging allows for longitudinal recordings of the same cells over several days, or even weeks , 29 in bThe ability of miniscopes to simultaneously assay at least 2 different emission/excitation spectra  enables The relative ease of recordings, the inexpensive nature of miniscope systems, and consequently their widespread use in many laboratories world-wide, is fueling the engineering of new sensors with the required signal to noise ratios. Although miniscopes have been almost exclusively used for imaging changes in calcium concentration in targeted cells, such as neurons and astrocytes, new molecular sensor developments (see below) promise to expand this repertoire.Recent advances in imaging go well beyond miniscopes and include other major advances which employ larger microscopes and head-fixed animals. The two-photon mesoscope is a powerful tool that allows an unprecedented number of neurons to be imaged at once . The keyHolographic two-photon imaging can combine simultaneous imaging and selective optogenetic manipulation by taking advantage of multiple wavelengths. Importantly, this technique can specifically target voxels of the imaging field in a three-dimensional, volumetric manner to precisely manipulate and image individually identified neurons , 33. TheA recent revolution in molecular sensors and reporters has transformed the field of molecular imaging, and these powerful tools may very well be one of the foundation stones of molecular systems neuroscience. Fluorescence, autofluorescence and bioluminescence have been used with a plethora of different kinds of molecular reporters and sensors to explore molecular structure and function in an unprecedented manner. This revolution in molecular imaging is based on two complementary strategies: imaging of molecular structures and events by tagging them with photon emitting reporters, and the visualization of molecular mechanisms (e.g. receptor activation) by taking advantage of fusions with specific molecular sensors.To visualize specific molecules, reporters such as fluorescent or bioluminescent proteins  are added so that they behave like “GPS devices” that track the movement of specific molecules in cells and circuits. Importantly, these tags are inert, and have a minimal impact on the endogenous properties of the targeted molecules, such as their activity, interaction with other molecules and movement in and out of cells. To precisely detect the subcellular distribution of a specific molecule by using just fluorescent tags like GFP, localization specific fluorescent tags have been developed. For example, the pH-sensitive GFP pHluorin can switch between a non-fluorescent state (quenched state) and a fluorescent state in response to changes in pH . Fusing 2+ imaging and engram identification to track engram activity [Since most GFP tagged proteins are continuously expressed, temporal information about the reported molecular events can be uncertain. To enable labeling within a specific time window, photoactivatable fluorescent proteins (PA-FPs) were developed , 41. Theactivity . FuelledTo observe molecular events in real time, sensors need to become brighter or change to a different fluorescent state immediately after such events occur (e.g. within milliseconds to seconds). To view G protein-coupled receptor (GPCR) activity, for example, a circularly permuted green fluorescent protein (cpGFP) was inserted at specific locations between the transmembrane helix 5 (TM5) and 6 (TM6) of the target GPCR Fig. a; cpGFP Tools based on resonance energy transfer (RET) have often been used to visualize protein–protein interactions. RET is the principal mechanism for intermolecular or intramolecular redistribution of electron energy following molecular excitation . BrieflyDespite the remarkable achievements made possible by FRET technology, FRET sensors have encountered several challenges in in vivo imaging. These include the low sensitivity and efficiency of FRET sensors which limit in vivo applications, the need for two different fluorescences for donor and acceptor for a single signaling event, thus limiting the number of events detected. By utilizing dimerization-dependent fluorescent proteins . Importantly, there are a number of novel molecular approaches that can be used to manipulate specific molecular mechanisms in specific cells and systems, and at specific times. These approaches are especially important in testing hypotheses derived from the reporters and sensors just described (and vice-versa). As always, the combination of diverse manipulation, observation and modeling tools is critical to confirm and understand any new biological finding.Historically, molecular manipulation tools have their roots in pharmacology  and genetic approaches . Over time these approaches have become more and more specific, and therefore more easily applicable to systems neuroscience questions. For example, region specific drug infusions through cannulae and osmotic pumps allowed targeting of drugs to specific brains areas, while conditional knock outs, the use of cell-specific promoters and viral vectors dramatically enhanced the spatial and temporal specificity of genetic approaches. More recently, light-mediated modulation of specific molecular systems has revolutionized our ability to manipulate molecular pathways in specific cells of targeted circuits and at times of our choosing. These developments have had a key role in bringing molecular approaches to systems neuroscience. Optical manipulation tools, for example, have been developed to target almost every step of cellular signaling events, from ligand binding, receptor activation, associated molecular signaling, all the way to downstream gene expression, and protein translation. Importantly, these molecular manipulations tools are perfect complements to the molecular reporting tools described above. Convergent findings with manipulation and reporting tools are key for discoveries in molecular systems neuroscience. Below, we will review some of the key approaches in the fast-evolving field of molecular manipulations tools which we propose will have an incalculable impact in systems neuroscience.Receptor activation is the first step of many molecular signaling pathways. Receptors play essential roles in translating extracellular signals into intracellular signaling events, and therefore they provide a key site to manipulate cell systems. Natural ligands, as well as agonists and antagonists, have been traditionally used to specifically modulate receptor activity and study related mechanisms. Unfortunately, these classic pharmacological tools cannot be selectively targeted to specific cells in a circuit, and their temporal resolution is limited by the time required for drug diffusion and degradation, making them difficult or impossible to use in system neuroscience studies. Below, we will review ground-breaking new molecular tools that circumvent these limitations, and therefore bring molecular manipulations to the purview of systems neuroscience.Optical modulation combined with sophisticated genetic approaches have made it possible to regulate receptor function in specific cells of targeted systems and in defined brain regions. Receptors for neurotransmitters, such as glutamate, GABA (Gamma-aminobutyric acid), dopamine, serotonin and noradrenaline have played a central role in neuroscience. Thus, the novel optical tools modulating the activation of these receptors provide ground-breaking opportunities to explore hidden properties of related systems with spatially and temporally precise receptor manipulations.Since most receptors need their ligands for activation, temporal and spatial regulation of ligands has been a key strategy to regulate receptors. Optopharmacological approaches include a number of photosensitive reagents that act on a wide range of molecules, including channels and receptors .Caged ligands, whose release is controlled by light, were a key developing milestone in the field. Caged molecules have photolabile protective moieties that cage the molecule in question, and keep it from interacting with its partners . Light activation removes the molecular cage, enabling the uncaged ligand to bind to its receptor. When caged, the compound/ligand stays inert until illuminated by the activating light wavelength. Uncaging following illumination, can trigger a rapid increase in the concentration of a specific ligand, therefore resulting in the prompt activation of a given receptor. Since uncaging is controlled by light, it can be restricted to specific sites and cells of targeted circuits. Furthermore, with two-photon light sources, uncaging can be directed to very specific sub-cellular sites, such as dendritic spines, thus significantly enhancing the spatial and temporal specificity of molecular manipulations . Current+ channels [2+ channels [Photoswitchable ligands are another key class of light controlled ligands. These ligands allow for bidirectional regulation (activation/deactivation) with precise time control. Unlike caged compounds, photoswitchable ligands can reversibly alternate between an active and an inactive state in response to two different wavelengths of light , and havchannels , 84 and channels .Uncaging as well as photoactivated ligands have been widely used for more than a decade to study synaptic transmission, compartmentalization of vesicles and receptors at the synapse, as well as synaptic plasticity . It is aTo avoid off-target effects, it is critical that caged and photoswitchable ligands are inert before activation, since high concentrations are always needed to ensure sufficient levels of these ligands around the targeted receptors. To address this challenge, photoswitchable ligands have been covalently tethered to receptor binding pockets, which result in very high concentrations near the sites of intended action , but comparatively lower concentrations elsewhere . PhotoswEfforts to control the function of receptors in real time have also taken advantage of hybrid receptors that incorporate, for example, the extracellular domains of opsins (so that the receptors become light activated) and that preserve the effector intracellular domains of the receptor. For example, since GPCRs share conserved structure, the extracellular loops of several GPCRs have been replaced with those of rhodopsin so that, upon light stimulation, the structure of the transmembrane domains of rhodopsin changes, thus activating downstream G protein complexes normally associated with the targeted GPCR . This ap+-channel  between an open or closed state [Ion Channels and other proteins can also be regulated by linking them to a class of proteins that changes conformation in response to light Fig. . These ped state . LOV 2 aed state –108). Sied state , 110. Oped state . An Optoed state .Fig. 3CoCLICR (Clustering Indirectly using Cryptochrome 2), which again uses engineered Arabidopsis cryptochrome 2, is a modular approach designed to allow optic control of the clustering of a range of transmembrane receptors . This isAlthough many of the methods mentioned in this section have already been used in neuroscience experiments, they all have specific individual disadvantages that somewhat limit their application. For example, caged ligands, as well as photoswitchable ligands, require that the ligand be exogenously supplied. Unless the ligand can be expressed by cDNA viral vectors at the desired levels, the high dosages required for these experiments can sometimes result in off-target effects  which coSimilarly, although the OptoXR approach can be widely used to tightly manipulate GPCR function, their highly conserved structures and signaling mechanisms do not always represent the diversity of extracellular and transmembrane domains seen in endogenous GPCRs. For example, some GPCRs form homodimers or heterodimers, and this can significantly change their downstream signaling, thereby affecting the precision and usefulness of the OptoXR approach –116. SimIn addition to receptor function, there are several signaling molecular mechanisms that can be manipulated for the study of cells and systems. These include protein interaction, phosphorylation, cleavage, aggregation/disaggregation, allosteric changes, etc. A number of different optogenetic tools have been designed to manipulate specific aspects of these signaling mechanisms. Next, we will review some examples that illustrate the enormous creative potential of these approaches.Protein–protein interactions (PPI) are critical for intracellular signaling. These interactions are required to form complexes that perform particular functions . For exTo control protein–protein interactions optogenetically, several Light-Inducible Dimerization (LIDs) systems have been developed that take advantage of light-sensitive domains including LOV2, Cryptochrome 2 (CRY2)- Cryptochrome-interacting basic-helix-loop-helix CIB1), Phytochrome (Phy)- Phytochrome-interacting factor (PIF) and Deinococcus radiodurans bacterial phytochrome  channels, and successfully demonstrated for the first time that inducing Ca2+-selective signaling in excitatory neurons of the hippocampal CA1 region is capable of enhancing contextual fear memory in mice [2+ modulators, termed monSTIM1 (monster OptoSTIM1), that show ultra-sensitivity to blue light [E281A) that exhibits stable basal intracellular Ca2+ concentrations (regardless of protein expression levels) and drastically enhanced photosensitivity when coupled with a superior CRY2 clustering system (CRY2clust). With MonSTIM1 it was possible to induce Ca2+ signaling in excitatory neurons or astrocytes in the cortex, hippocampus and thalamus of awake mice with non-invasive light simulation. When applied to the anterior cingulate cortex (ACC) of the mouse brain, monSTIM1 effectively enhanced short-term (4 min) and long-term (24 h) memory for observational fear in mice. This experiment directly demonstrated how the fine tuning of Ca2+ signaling in a single brain region can effectively affect memory encoding.Intracellular Ca in mice . They alue light . Using sIn addition to controlling dimerization/aggregation, optogenetic strategies have also been focused on controlling enzyme activity by inducing light-triggered changes in protein conformation that result in activity changes in targeted proteins. For example, light induced structural changes in AsLOV2 were used to change the activity of Ras-related C3 botulinum toxin substrate 1, Rac1 (PA-Rac1), a small signaling GTPase involved in a diverse array of cellular functions. Briefly, the complete LOV2-Jα sequence was fused to the amino terminus of a constitutively active Rac1. Before light activation, the LOV domain tightly binds the Jα helix domain, and this prevents Rac1 effectors from binding to Rac1. Light stimulation unwinds the Jα helix, which releases steric inhibition of Rac1, allowing this protein to bind to its effectors . AmongstOther than controlling intracellular signaling, opto-tools have also been engineered to modulate gene expression. Some of these tools take advantage of transcription systems such as Cre/Flippase (Flp) recombinase or tetracycline system, that have been used to control the expression of target genes under the control of the artificial promoters of these systems, while others were designed to directly bind targeted promoters and thus modulate the expression of the associated genes. Site specific recombinase systems, such as Cre/locus of X-over P1 (Loxp), Flp/Flippase recognition target (FRT), are widely used for cell-type specific gene modulation by expressing these systems in transgenic mice or by delivering them with viral vectors . AlthougNuclease-dead Cas molecules (dCas9 and dCas12a), that still bind DNA guided by single guide RNA (sgRNA), have been fused to light induced dimerizers , such that light stimulation triggers the expression of targeted genes. RNA based targeting makes this system exquisitely specific. Similar strategies, in which light induced dimerizers were fused to specific transcription factors, have also been used to optogenetically control the transcription of target genes –153. SinAnother strategy to optogenetically control gene transcription takes advantage of cellular mechanisms that guide transcription factors to the cell nucleus where they modulate gene expression. Briefly, these mechanisms involve small protein segments that function as cell nuclear export signals (NES) and nuclear localization signals (NLS) to control the cellular localization of specific transcription factors . For exaSubcellular localization of RNA also plays a key role in the function of neurons, such as the targeting and local translation of RNA at synapses. A CRISPR/Cas9 derived system (RNA-targeting Cas9 or RCas9) has also been used not only to visualize the trafficking of specific mRNAs in cells (with EGFP fusions), but also to optogenetically modulate the trafficking of specific RNAs by trapping (or releasing) these mRNAs in molecular clusters within the cell . Functio), proteins ten times smaller (~ 15 kDa) than an antibody, have comparable affinities for their antigens but are highly soluble, stable, and have excellent tissue penetration, thus making them useful as modulation tools for protein function [The ability of monoclonal antibodies to very specifically target their antigens has been widely used in research and in medical treatments (e.g. cancer therapy). However, their large size (150 kDa), and non-covalently associated variable domains, make it essentially impractical to express such large proteins with cDNA constructs, limiting their usefulness for many applications, including systems neuroscience. In contrast, nanobodies  can be used to inactivate DNA, RNA, lipids and proteins. With Chromophore-Assisted Laser or light Inactivation (CALI), it is possible to optogenetically produce ROS and inactivate a number of intracellular molecules in cell sites targeted by light. Because the free radical species generated during CALI are short-lived (less than 1 s), and light can be limited to small subcellular regions, this approach is an important spatially and temporally controlled loss-of-function tool , 167. AlThe development of opto tools has been centered on two key aspects: (1) better spatial and temporal specify and (2) novel combinations of molecular systems, which continue to open novel modulation opportunities.Although optogenetics enable the modification of neuronal activity with high spatial and temporal resolution, there are limitations that need to be addressed. For example, light delivery often involves the implantation of optic fibers, and this inevitably results in some damage to the targeted tissue, especially when deep brain regions are studied , or when multiple-areas need to be modulated. Deep tissue penetration by light is another challenge that needs to be addressed. Opsins with red-shifted spectra have been developed to allow possible light delivery through the skull and to deeper brain areas , 172. AnBioluminescence involves the emission of light as a result of a cellular biochemical reaction. In contrast to fluorescence and phosphorescence, bioluminescence reactions do not require exogenous light activation. Instead, in bioluminescent systems the excitation light is produced through the oxygenation of a substrate, generically called luciferin, and the activity of the enzyme luciferase –175: a lAlthough bioluminescent approaches suffer from inferior temporal resolution , the spatial resolution of these systems can be superior because illumination comes from light sources with cellular and even molecular resolution. Luciferase can be expressed in any target cellular/subcellular region, and only the nearby opsins receive enough photons to be activated. Therefore, bioluminescent derived systems allow the investigation of questions that require optogenetic modulation at higher spatial resolutions. For example, luciferase has been combined with the SPARK2 system, which can label beta2 adrenergic receptor activation, so that only cells closely interacting with the luciferase expressing cells are activated .Another limitation of current optogenetic systems is that the enhancement and inhibition of molecular/cellular activity is done arbitrarily at a time chosen by the experimenter, and not as a result of some molecular, cellular or circuit event intrinsic to the systems being studied. To circumvent this limitation, molecular sensors have been combined with optogenetic modulators so that specific biological phenomena perceived by the sensor triggers when optogenetic activation takes place. For example, naturally occurring neural dynamics during behavior can provide the trigger for subsequent precisely timed optogenetic manipulation to mimic or block endogenous activity. GCaMP6 based fibre photometry was used to identify differences in NAc D2 dopamine receptor-expressing neuronal activity during the decision process. Optogenetic stimulation was applied only during the decision period to mimic endogenous dynamics of single-trial control of risk-seeking behavior , 180.As an extension of this idea, the promoter of the activity dependent immediate early gene c-Fos has been engineered to control the transcription of a light controlled tTA (opto-tTA) so that the transcription of specific genes is both activity and light-dependent. Importantly, unlike conventional tTAs , the regAnother exciting new tool involves a GPCR-coupled CRISPR-Cas system. This system can sense the activation of GPCRs, through the TANGO or Chacha systems described above. Then, this results in the uncaging of dCas9, which goes on to modulate gene expression . In the The incredible success of systems neuroscience is in no small part shaped by the foundational methods of the field, including electrophysiology and modeling. It is absolutely remarkable how far the field has come despite the fact that the majority of studies were focused on one group of phenomena  of a single class of cells in the brain (neurons), and they were carried out with different variants of a single experimental approach (electrophysiology). This success is all the more remarkable since we now have overwhelming evidence that the complexity of behavior derives from nearly every cell type in the brain, and can be traced back to a wide range of biological mechanisms that go well beyond neuronal firing dynamics. Nevertheless, the majority of ideas and models in systems neuroscience to this day remain firmly entrenched in the illustrious history of the field, in no small part due to the enormous creativity and generative potential of the ideas that serve as its foundational stones. Despite this, it is now time to look into the future of systems neuroscience, a future that will be firmly grounded within its enormously successful past without being restricted by it. The limited sample of exciting technological developments that we briefly reviewed here suggest that it may be worthwhile to leverage these powerful techniques to reimagine the approaches and questions asked in the field. Like many others in the field, we suspect that attempting to understand systems only in molecular terms would be like studying art by investigating the molecular properties of the materials used. This would not only be shortsighted, but it would once again circumscribe the future of the field in a narrow conceptual space. Our view is that imaging and manipulating the very molecular properties that both shaped brain systems  during evolution, and that now account for and constrain much of their functionality, will open countless windows into the properties and mechanisms of the systems  that underlie the marvelously perplexing complexity of brain states and behavior.