Oppositional defiant disorder (ODD) is a major mental health concern and highly prevalent among children living in poverty-impacted communities. Despite that treatments for ODD are among the most effective, few children living in poverty receive these services due to substantial barriers to access, as well as difficulties in the uptake and sustained adoption of evidence-based practices (EBPs) in community settings. The purpose of this study is to examine implementation processes that impact uptake of an evidence-based practice for childhood ODD, and the impact of a Clinic Implementation Team (CIT)-driven structured adaptation to enhance its fit within the public mental health clinic setting.This study, a Hybrid Type II effectiveness-implementation research trial, blends clinical effectiveness and implementation research methods to examine the impact of the 4Rs and 2Ss Multiple Family Group (MFG) intervention, family level mediators of child outcomes, clinic/provider-level mediators of implementation, and the impact of CITs on uptake and long-term utilization of this model. All New York City public outpatient mental health clinics have been invited to participate. A sampling procedure that included randomization at the agency level and a sub-study to examine the impact of clinic choice upon outcomes yielded a distribution of clinics across three study conditions. Quantitative data measuring child outcomes, organizational factors and implementation fidelity will be collected from caregivers and providers at baseline, 8, and 16 weeks from baseline, and 6 months from treatment completion. The expected participation is 134 clinics, 268 providers, and 2688 caregiver/child dyads. We will use mediation analysis with a multi-level Structural Equation Modeling (SEM) , as well as mediation tests to examine study hypotheses.The aim of the study is to generate knowledge about effectiveness and mediating factors in the treatment of ODDs in children in the context of family functioning, and to propose an innovative approach to the adaptation and implementation of new treatment interventions within clinic settings. The proposed CIT adaptation and implementation model has the potential to enhance implementation and sustainability, and ultimately increase the extent to which effective interventions are available and can impact children and families in need of services for serious behavior problems.NCT02715414. Registered on 3 March 2016.ClinicalTrials.gov, ID: The online version of this article (doi:10.1186/s13063-017-2331-7) contains supplementary material, which is available to authorized users. Oppositional defiant disorder (ODD) is a chronic and impairing mental health disorder that afflicts between 1 and 21% of the population, and primarily elementary school-age children, with the median age of onset being 11.6 years [Family poverty is one of the greatest risk factors for the onset and perpetuation of DBDs –4: studiTreatments for DBDs are among the most well-studied and efficacious , yet as The 4Rs and 2Ss Multiple Family Group (4Rs 2Ss MFG) for Strengthening Families Program is an evidence-based, manualized, family based treatment for families of children with disruptive behaviors –15. ReceThe 4Rs and 2Ss MFG intervention draws from group therapy techniques, systemic family therapy, and behavioral parent training . It was Although positive outcomes from the 4Rs and 2Ss MFG were found in prior studies, significant implementation challenges emerged . OngoingThere has been a growing interest within the mental health field in implementation processes that facilitate the uptake and adoption of evidence-based practices in community-based settings , 20. TheHybrid effectiveness-implementation research designs were developed to focus both on an intervention’s effectiveness and implementation processes, either sequentially or simultaneously, which is proposed to accelerate the process to integrating EBPs in real-world practice settings . As noteThis study reflects a combination of clinical effectiveness and implementation research methods, with aims focused on replicating child outcomes associated with the 4Rs and 2Ss MFG, and gathering information about family and clinic/provider-level moderators of implementation. A secondary objective is to test an innovative method of adaptation of the 4Rs 2Ss MFG through the formation of change teams that are hypothesized to enhance outcomes and inner contextual factors. Within the academic literature on implementation processes, there is a growing emphasis upon staff serving as champions or change agents to implement EBPs and address potential barriers to adoption , 29, 35.The short-term and longitudinal impact of the 4Rs and 2Ss MFG model on child ODD and functioningFamily level mediators, such as parenting and family processes, that impact child outcomesClinic- and provider-level factors  upon implementation of the 4Rs and 2Ss MFG implementation, andThe impact of Clinic Implementation Teams on clinic- and provider-level moderators of 4Rs and 2Ss MFG implementation and integrationIn 2014, the National Institute of Mental Health (NIMH)-funded “Family Groups for Urban Youth with Disruptive Behavior.” The grant began with a planning stage in 2015 and will be completed by 2019. The four aims of the study are to examine:All NYS Office of Mental Health (OMH) child-serving public outpatient mental health clinics across the five boroughs of New York City (NYC) are eligible to participate. To secure the sample of clinics, the study team obtained a master list of 168 clinics from OMH. Based on prior statewide studies conducted by the investigative team, it was estimated that approximately 80% of the clinics would participate; thus, we estimated that approximately 134 clinics will enroll in the study.n = 896 per arm, or 2688 total), and two providers per arm, yielding a total of 268 providers. Researchers will consecutively consent families to participate in the study until a sample of 20 caregiver/child dyads per clinic is achieved. Eligible caregivers are 18 years of age or older, English or Spanish speaking, and the primary caregiver of a 7- to 11-year-old child with ODD. Eligible providers are staff members who work in a participating clinic in a clinical capacity, and are English speaking.At each clinic, we anticipate enrolling 20 caregiver/child dyads  single-site  multi-site agencies with between two to four clinics and, (3) mega-agencies that had five or more clinics. Each list was organized alphabetically.A separate randomization process was employed for each list. Clinics within single-site agencies were stratified by borough, and a random number generator was employed to randomly assign clinics to one of three conditions: (1) condition 0, which was services as usual (SAU), (2) condition 1, the 4Rs and 2Ss MFG, and (3) condition 2, the 4Rs and 2Ss MFG + CIT . Once the initial number was generated, subsequent clinics were assigned the next number in the sequence . Of note, the research staff member was unaware what numbers corresponded with which condition . This procedure was undertaken in order to minimize allocation bias.Multi-site agencies with between two and four clinics were also organized alphabetically within borough, and a random number generator was used to assign the initial clinic to a particular condition. Moreover, additional clinics that were part of the agency in which the initial clinic was randomized received the same condition. For example, if one clinic was assigned condition 0, the remaining clinics, regardless of which borough they were located in, were also assigned to condition 0.Finally, agencies with five or more clinics were given the choice to select the study condition in which they would participate with an even distribution of these mega agencies occurring across the three study conditions.The 4Rs and 2Ss Multiple Family Group interventionThe 4Rs and 2Ss MFG is a 16-week group for urban children of between 7 and 11 years of age with ODD and their caregiver(s). In this model, six to eight families meet in weekly sessions focused on addressing family and parenting factors associated with behavior problems –33. SessThe 4Rs and 2Ss MFG + CITIn addition to implementing the model and receiving supervision, a CIT consisting of providers, a clinic director and supervisor will participate in ongoing meetings with the research team’s adaptation specialist for a guided analysis of the structure, content and processes of the treatment model, including those that are amenable to modification and those that must remain unchanged in order to maintain fidelityStandard careStandard care includes the services that the clinic normally provides to children with behavioral problems, including outpatient mental health therapy and pharmacologyThe study consists of three arms: two active treatment and one comparison condition:Group facilitators will provide supervision by phone on a bimonthly basis, and three face-to-face supervision meetings after each fidelity check. Supervision will be conducted by clinical staff who have extensive knowledge of the 4Rs and 2Ss model, and who have supervised clinicians in other initiatives using this model. Additionally, two independent raters will conduct fidelity checks for a minimum of three sessions to ensure adherence to the treatment model. As fidelity checks will be conducted by two personnel, inter-rater reliability will be computed. Providers falling below 80% in fidelity will be given additional supports, such as additional supervisory sessions, to improve adherence.Of the approximately 134 clinics that will be invited to participate; 37 clinics have been enrolled in the study thus far. Data collection has begun at 14 clinics in the SAU clinic, eight clinics in condition 1 and seven clinics in condition 2. Others have agreed to participate and data collection will begin shortly.Data collection will occur at baseline, 8, and 16 weeks from baseline, and at 6-month follow-up. Demographic characteristics about familial factors  will be collected via a questionnaire used in prior studies. A separate demographic questionnaire will be administered to providers to gather information including their age, race/ethnicity, education, and credentials.α at baseline of .70 and .89, respectively [will be measured by the Disruptive Behavior Disorders Rating Scale Oppositional Defiant Subscale . Completectively .α at baseline of .86 [Child ODD will also be measured by the Iowa Conners Rating Scale-Oppositional/Defiant Subscale (IOWA CRS OD) , a widele of .86 .Child functioning will be measured via the Impairment Rating Scale (IRS),  a six-itα of above 0.8 [Caregiver depression will be measured using the short form of the Center for Epidemiologic Studies Depression Scale (CESD) -46, a frbove 0.8 α values reported as baseline, mid test, post test, and 6-month follow-up, were 0.91, 0.92, 0.94, and 0.94, respectively [The Parenting Stress Index (PSI) short form will be administered to measure stress . This 36ectively .α ranging from 0.59 to 0.84 in the first study and 0.80 to 0.92 in the second study [The Alabama Parenting Questionnaire (APQ-9) short form will measure parenting factors, and specifically positive parenting, inconsistent discipline, and poor supervision . This scnd study .α of 0.72 (Roles) and 0.75 (Communication) [Two subscales of the McMaster Family Assessment Device (FAD)  will meaication) .To measure social support, the family subscale of the Multidimensional Scale of Perceived Social Support (MSPSS)  will be α = 0.85 for the Family subscale) [The MSPSS has strong factorial validity as well as good internal and test-retest reliability  .Attendance will be collected, and a total number of sessions attended will be tallied for each participant.These will be assessed using the Kazdin Barriers to Treatment (KBT) scale , which iappeal of EBP, the likelihood of adopting EBP in light of such requirements, openness to new practices, and the degree to which divergence is perceived between usual practice and research-based/academically developed interventions. Five possible responses are provided on Likert scale, from 0  to 4 (to a very great extent). For each subscale, a mean score is calculated and the fourth (divergence) is reverse coded. A higher mean score indicates a greater degree of the construct reflected by that subscale. A study using this scale to assess service provider attitudes from 51 mental health service programs found good internal consistency reliability, with an overall Cronbach’s of 0.77 [The Evidence-Based Practice Attitude Scale  will ass of 0.77 .α of above .70 [This will be assessed via the Texas Christian University (TCU) Organizational Readiness for Change (TCU ORC) instrument . Responsbove .70 .α values ranging from .61 to .83 [The Measure of Beliefs about Participation in Family Centered Service Delivery  will ass1 to .83 .Fidelity monitoring tools will assess facilitator adherence to curriculum content  and clinical skills . These fidelity assessments were standardized during fidelity observations of prior 4Rs and 2Ss MFGs studies , 18.These include size, fiscal health and funding streams, and populations served will be analyzed from two pre-existing datasets collected by New York State Office of Mental Health (NYSOMH) about organizational composition and service delivery.Aim #1: to examine the short-term and longitudinal impact of 4Rs and 2Ss MFG on child ODD and functioningHypothesis #1: youth who participate in 4Rs and 2Ss MFG will display significantly reduced conduct difficulties and increased functioning over time compared to those involved in standard care: youth in the 4Rs and 2Ss MFG + CIT condition will evidence the greatest magnitude of change in outcomes over timeAim #2: to examine family level mediators  of child outcomesHypothesis #1: based on existing 4Rs and 2Ss MFG findings, specific family level variables  will be associated with significantly greater impact on child outcomes over time relative to social support, established responsibilities, and respectful communicationAim #3: to examine clinic-  and provider-level moderators  of 4Rs and 2Ss MFG implementation and integrationHypothesis #1: leadership support will evidence significantly greater impact on 4Rs and 2Ss MFG implementation/integration in comparison to general readiness and clinic climateHypothesis #2: provider motivation will evidence significantly greater impact on 4Rs and 2Ss MFG implementation/integration relative to other provider-level variablesAim #4: to examine the impact of CITs on clinic- and provider-level moderators of 4Rs and 2Ss MFG implementation and integrationHypothesis #1: clinic directors assigned to MFG + CIT will evidence significantly higher readiness for innovation and support, and providers assigned to MFG + CIT will evidence significantly higher preparedness and motivation to implement MFG relative to MFG aloneHypothesis #2: preparedness, motivation of providers, and leadership support assigned to MFG + CIT will be maintained over time and be significantly higher relative to MFG aloneHypothesis #3: providers assigned to MFG + CIT will evidence significantly enhanced fidelity to MFG relative to MFG onlyHypothesis #4: clinics assigned to MFG + CIT will have more MFGs initiated and completed (with high fidelity and attendance) relative to MFG aloneThe purpose of this study is to examine outcomes associated with the 4Rs and 2Ss MFG, implementation processes that impact uptake and sustained use of the intervention over time, and the impact of an innovative change agent team upon outcomes. The following hypotheses will be tested:Overall, the two primary outcomes being tested in this study are changes in child behavior and child functioning. These outcomes are being tested specifically in aims 1 and 2, as measured by the Disruptive Behavior Disorders Rating Scale Oppositional Defiant Subscale , Iowa CoThe overall analytic structure uses a three-level, multi-level Structural Equation Modeling (MSEM) framework with family level variables at level 1, provider variables at level 2, and clinic variables at level 3. The analytic structure is complex, so we highlight our approach using Fig. We will employ mediation analysis with two-level MSEM models as explicated by Preacher, Zhang and Zyphur  and exteThe overall analytic structure requires strategic use of different dummy contrasts and limited information estimation of various MSEM models that map onto the questions dictated by the aims, coupled with judicious use of mediational tests linked to joint significance testing. An alternative analytic structure is to work with all four time periods simultaneously in a classic growth curve model and then to model the linear growth in variables as a function of the provider and clinic variables in strategically defined limited information estimation frameworks dictated by the overall model structure in Fig. A Data Monitoring and Review Board is responsible for reviewing this study’s data to ensure quality and integrity, the safety of participants and study progress, and to make any determinations regarding potential changes to the study. This is comprised of the investigative team with ongoing support from New York University’s Institutional Review Board (IRB). The DMRB meets on a quarterly basis under normal circumstances, or within two business days should an adverse event occur. This process is independent from the sponsors, although they will be notified in the case of a significant change to study protocol or in the case of an adverse event.There are potential but minor risks for providers associated with participating in the study, specifically, feeling coerced into participating, loss of confidentiality, and experiencing discomfort. These risks are addressed in several ways: potential participants are assured that participation is voluntary and will not affect their employment status, all study materials are coded with ID numbers and do not include identifying information, and all data will be maintained in password-protected computer files.Among caregivers, there is a risk that they may feel uncomfortable with some of the questions in the assessment measures. However, research staff clarify that they do not need to answer any question that makes them feel uncomfortable. The risk to loss of confidentiality is also present for caregivers and is addressed following the same precautions and guidelines used for protecting provider confidentiality. Finally, there is a potential risk that symptoms may worsen as a consequence of the progression of a serious mental health issue or response to a service provided. In the event that this happens, study participation will be halted and appropriate care provided immediately in accordance with clinic procedure.This study represents an innovative approach to examine the uptake and sustainment of a National Registry of Evidence-based Programs and Practices (NREPP)-approved, evidence-based treatment for child DBDs, and the organizational factors that impede or facilitate adoption of EBPs in public mental health settings. This study will also examine a novel approach to adapt an EBP in a structured way, in order to enhance its fit with the needs of the populations being served and the organizational context. One of the strengths of this study is that it will test uptake across the entire child-serving, public mental health service system in the five boroughs of NYC.The study aims to generate knowledge needed to address seemingly intractable urban service delivery challenges, including lack of engagement of low-income youth with serious DBDs and their families, too few clinics offering family based, evidence-informed services; lack of scalable, empirically supported interventions designed for resource-strapped child settings; and few empirically supported options for public policy-makers to support the uptake and integration of service innovations within behavioral health care.n = 792) of the sample of providers and caregivers have been enrolled. Clinic recruitment for MFG and MFG + CIT have been initiated, and some clinics have already completed training and have participated in adaptation meetings. Recruitment of clinics for the experimental arms will be ongoing until enrollment targets are met. Groups for both MFG and MFG + CIT are expected to begin in the first trimester of 2017. A populated Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) Checklist Additional file 2:Data Monitoring Committee Information. Information regarding the Data Monitoring Committee for this study. (DOC 15 kb)


Background: The exosome complex plays key roles in RNA processing and degradation in Eukaryotes and Archaea. Outstanding structural studies identified multiple pathways for RNA substrates into the exosomein vitro, but identifying the pathway followed by individual RNA speciesin vivo remains challenging.Methods: We attempted to address this question using RNase protection.In vivo RNA-protein crosslinking (CRAC) was applied to the exosome component Rrp44/Dis3, which has both endonuclease and exonuclease activity. During CRAC, the exosome was purified under native conditions and subjected to RNase digestion, prior to protein denaturation and cDNA cloning. The resulting high-throughput sequence reads were stratified by length of the cDNA sequence. This should reflect RNA fragment lengths, and therefore the RNA region that was protected by exosome binding. We anticipated major read lengths of ~30nt and ~10nt, reflecting the “central channel” and “direct access” routes to the Rrp44 exonuclease active site observedin vitro.Results: Unexpectedly, no clear peak was observed at 30nt, whereas a broad peak was seen around 20nt. The expected ~10nt peak was seen, and showed strong elevation in strains lacking exonuclease activity. Unexpectedly, this peak was suppressed by point mutations in the Rrp44 endonuclease active site. This indicates that the short fragments are degraded by the exonuclease activity of Rrp44, but also suggests that at least some may be generated by endonuclease activity.Conclusions: The absence of 30nt protected fragments may reflect obligatory binding of cofactors at the entrance to the exosome central channelin vivo. The presence of ~20nt fragments apparently indicates an access route not yet reported fromin vitro studies. Confident mapping of 10nt reads is challenging, but they are clearly derived from a subset of exosome targets. In particular, pre-rRNA species, which are major exosome targets, are strongly disfavored for the generation of short reads. Rrp44 is composed of an N-terminal PIN (PilT N terminus) domain with endonuclease activity, two continuous RNA-binding cold-shock domains (CSD domains), an RNB domain carrying the exonuclease active site, and an RNA-binding S1 domain . Initial (CRAC) . To alloin vivo is very challenging. These pathways involve distinct conformations of the exosome and would be expected to protect different lengths of the substrate RNA.In vitro analyses have confirmed the protection of the 3’ terminal 30-33 nt for RNAs threaded through the channel, whereas only ~9-10 nt might be expected to be protected on the direct access route. The aim of the work reported here was to use this distinction to identify RNA substrates for each pathway.RNAs that are targeted to the exonuclease domain of Rrp44 can follow at least two routes; threading through the central barrel of the exosome complex, or direct access to the active site. However, identifying the substrates that follow each of these pathways6 - TEV protease cleavage site – 2 copies of the Z-domain of protein A) , or lacked endonuclease activity, due to point mutations at each of the four conserved endonuclease active-site amino acids .CRAC was performed on a Rrp44 construct expressed from the endogenous locus and carrying a tripartite C-terminal HTP tag (Histein A) . OtherwiDuring CRAC analyses , bait prin vitro analyses, we expected two major length populations; around 30-33 nt from RNAs threaded through the central channel, and around 9-10 nt from RNAs that directly access the Rrp44 exonuclease site . Mapping of ~10 nt fragments therefore creates significant problems with false positive results. Despite this we were able to identify sites where the long and short read populations yielded very consistent mapping data. Prioritization of the data, such that ambiguous reads are first mapped to the most common exosome substrates, appeared to substantially improve the quality of mapping. This provided clear evidence that some substrates are strongly disfavored in the short reads. This was most marked for the pre-rRNAs, which are normally the predominant exosome substrate. Recent data indicated that the exosome-associated RNA helicase Mtr4 is actively and specifically recruited to pre-rRNAs  (RRID:SCR_005012). Rrp44 and Rrp44-exo CRAC datasets were previously published (GSE77863). Rrp44-exo split-CRAC and Rrp44-endo CRAC datasets were previously published in (GSE40046 andGSE94889).Most of the primary sequence data were previously published and deposited in NCBI Gene Expression Omnibus , grown in SD-medium to log phase and UV crosslinked  to covalently bind RNA to protein. Cells were lysed in buffer containing 50 mM Tris-HCl pH 7.8, 1.5 mM MgCl2, 150 mM NaCl, 0.1% NP-40 and 5 mM β-mercaptoethanol, and RNA-protein complexes were isolated by binding to an IgG column. Bound material was washed briefly in the same buffer, but with 1M NaCl  using Novoalign  with genome annotation from Ensembl (EF4.74) (EF4.74) , with paCounting overlaps with features and prioritization. Downstream analyses were performed using pyCRAC software  and normalized per reads per millions.GSE77863,GSE40046 andGSE94889.All sequence data are available from GEO (RRID:SCR_005012) under accession numbers The revised version has addressed most of the questions raised by the reviewers. Certainly new experiments can be performed in the future to reveal the exosome-RNA interactions more thoroughly.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The exosome complex is an important RNA degradation and processing machinery that is responsible to degrade RNAs from their 3’ ends. Previous works suggested the presence of at least two routes for RNA substrates to be recruited to the exosome complex’s exonuclease activity site in the Rrp44/Dis3 protein. The Rrp44 protein also has a weak endonuclease activity site in its N-terminal region. The activity of exosome is also regulated by a few co-factor proteins. These all lend the complex a rather complicated RNA degradation or processing mechanism that still awaits to be fully revealed. The authors in this work used CRAC technology in combination with the RNase protection to analyze the RNA substrates that are loaded to Rrp44 protein and protected by the exosome complex. Their results showed the accumulation of 10-nt RNA species bound to Rrp44’s C-terminal region and the absence of 30-nt RNA species. Instead, they observed a broad shallow peak around 20-nt. Interestingly, the lack of endonuclease activity caused the reduction of the 10-nt RNA species. Deep sequencing analysis of the bound RNAs showed a whole spectrum of RNA species bound to Rrp44, indicating a complex behavior of the exosome in RNA substrate recruitment in vivo. The authors proposed the presence of another pathway for RNA substrates to be recruited to the exosome and a potential role played by the endonuclease during the different pathways. This work provided new data to study the exosome-RNA transactions although the exact mechanism seemed more complicated than proposed previously or in this work. More data would be helpful if the authors can also perform similar CRAC of yeast strains lacking Rrp6 gene to understand the cytoplasmic exosome’s substrate more clearly. A minor issue is that the author didn’t explain what the Rrp44-exo-S1 mutant is.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. in vivo with the wild type exosome or with mutants that are defective for the exo- or endonucleolytic function.  They exploit the RNase protection step included in the CRAC protocol and assess the average length of protected fragments to infer the topology of RNA degradation by the exosome . Using a standard protocol for sequencing (50nt reads) they see a distribution of length with a clear peak at very short reads (6-8 in the wt and 9-10 in the exo- mutant). Formation of these short fragments requires the endonucleolytic function of the exosome. The authors propose that these fragments derive from endonucleolytic cleavage of stalled substrates in the central channel of the exosome.In this article the authors analyze the RNA fragments that associate Using a longer read sequencing protocol, they observed the presence of two additional peaks (39 and 44nt) that likely represent fragments derived from substrates that are being degraded after threading through the central channel. Mapping of these read classes allowed inferring on the substrate preferences for the mechanisms of degradation . This is a well-conducted study that provides many interesting details on the mechanism of RNA degradation by the exosome. The data convincingly support the model proposed. What follow are a few suggestions that could complement the study.The distribution of the mismatches due to crosslinking is not exploited in the study. This could provide useful information on the nature of the reads. For instance, the authors propose that the 39 and 44nt reads derive from protection either by the sole central channel, or by the central channel and the helicases associated (Mtr4 or Ski2). If this is true, the two families of reads should have an identical distribution of crosslinking sites when aligned to the 3’ end. Also, reads derived from threading or direct access should have a different distribution of mismatches.The authors propose that stalled substrates threaded through the central core are cleaved by the endo activity for further processing. This should imply endo cleavage of substrates within the central core. Is this consistent with current structural models ? The authors should comment on this.Use of a mutant that prevents threading might allow demonstrating that the 39nt and 44nt reads indeed derive from substrates being threaded through the central core  I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. in vitro experiments, one would predict that the exosome would protect RNAs of ~30 bases when they are threaded through the barrel of the exosome, and ~10 bases when the RNA accesses the active site directly.This manuscript addresses the paths by which RNAs enter the exonucleolytic and/or endonucleolytic active sites of the Rrp44, which is the catalytic subunit of the eukaryotic exosome. This is an interesting issue since the exosome has two main nucleolytic activities and their relative importance and how different RNAs access these different sites is not clear. Based onin vivo.  The approach is to cross link RNA to the exosomein vivo, purify the exosome and then the cross-linked RNAs are digested to protected fragments subjected to deep sequencing.  This work revealed the WT exosome protects a broad range of RNA sizes with three main classes: 1) an enrichment ~6-8 bases, which is dependent on the endo activity, and converts to ~10 bases in the absence of exo activity: 2) a broad size distribution from 12-34 that is largely independent of exo or endo nuclease activities, and 3) RNAs of 39 and 44, which are not dependent on the direct entry channel and are likely to reflect RNAs that are threaded through the exosome barrel, but more than 30 bases are protected possibly due to exosome co-factors. The main contribution of this work is to use deep sequencing to identify fragments of RNA protected by the exosome in vivo will suggest unanswered questions about how RNA molecules interact with the exosome and access the active sites. Taken together, this provides insight that the endonuclease activity may be quite important for releasing RNAs that are stalled during exonucleolytic degradation.  It also highlights that understanding the size of RNAs protected by the exosome The experiments are well done and the interpretations valid.  Some suggestions that could be considered to improve the work are given below:  Although not required for indexing, it would be interesting to know if the reads that map to mRNAs  map to distinct subsets of mRNAs. For example, the exosome would be predicted to preferentially target mRNAs with slow decapping rates, which might be revealed by a more granular analysis of the read distribution to different mRNAs.I might rephrase the summary of the work to highlight that there is a clear set of fragments of 39 & 44 bases, which would be consistent with RNAs threaded through the barrel of the exosome but protected by additional co-factors.  As written now, one is not aware of these fragments till deeper in the manuscript and the comments about co-factors in the conclusions come a bit out of the blue.Although not required for indexing, it would be interesting to see how the shorter reads are affected by the exo-S1 mutations.   I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


The postsynaptic density (PSD) contains a complex set of proteins of known relevance to neuropsychiatric disorders such as schizophrenia and bipolar disorder. We enriched for this anatomical structure in the anterior cingulate cortex of 16 bipolar disorder samples and 20 controls from the Stanley Medical Research Institute. Unbiased shotgun proteomics incorporating label-free quantitation was used to identify differentially expressed proteins. Quantitative investigation of the PSD identified 2033 proteins, among which 288 were found to be differentially expressed. Validation of expression changes of DNM1, DTNA, NDUFV2, SEPT11 and SSBP was performed by western blotting. Bioinformatics analysis of the differentially expressed proteins implicated metabolic pathways including mitochondrial function, the tricarboxylic acid cycle, oxidative phosphorylation, protein translation and calcium signaling. The data implicate PSD-associated proteins, and specifically mitochondrial function in bipolar disorder. They relate synaptic function in bipolar disorder and the energy pathways that underpin it. Overall, our findings add to a growing literature linking the PSD and mitochondrial function in psychiatric disorders generally, and suggest that mitochondrial function associated with the PSD is particularly important in bipolar disorder. Synaptic plasticity is implicated in neuropsychiatric disorders,5 and thus the PSD is implicated.13 However, although known constituents of the PSD have been implicated in bipolar disorder and schizophrenia at both gene16 and protein expression levels,20 no major findings are reported to contribute to bipolar disorder risk.22 As synaptic plasticity is highly dependent on mitochondrial function,23 energy metabolism acting at the level of the PSD may underpin PSD dysfunction in bipolar disorder and indeed other neuropsychiatric disorders.The postsynaptic density (PSD) is a highly organized structure attached to the postsynaptic neuronal terminal. It comprises a complex network of cytoskeletal scaffolding and signaling proteins that facilitate the movement of receptor and signaling complexes. The PSD is critical for normal neurotransmission and synaptic plasticity24 The differential expression of the PSD in schizophrenia compared with controls was first reported recently by our team25 highlighting altered pathways of endocytosis, long-term potentiation and calcium signaling in schizophrenia. The identified PSD proteome  was significantly associated in gene set enrichment analysis with schizophrenia, validating independent reports of PSD enrichment in schizophrenia. Furthermore, numerous mitochondrial proteins were differentially expressed in the PSD schizophrenia proteome and these changes were not associated with antipsychotic administration.25 This was in keeping with previous studies of the brain in neuropsychiatric diseases.36 These findings are of relevance to bipolar disorder, considering the known overlap with schizophrenia in terms of clinical presentation, genomic, structural imaging,37 transcriptome and protein expression.26 To date, no study has assessed the protein expression of the PSD in the cortex in bipolar disorder compared with controls.Mass spectrometry-based proteomic methods have the ability to reliably identify and quantify several thousands of disease-associated protein changes derived from complex anatomical structures. The reliable quantitation of low-abundance proteins within specific cellular compartments until recently has been challenging and this has led to a shift in the use of pre-fractionation enrichment methods combined with mass spectrometry-based proteomic techniques. This approach has successfully yielded a detailed characterization of the PSD proteome in rodents and in healthy postmortem human brain tissue.41 We undertook a label-free liquid chromatography–mass spectrometry (LC-MS/MS) investigation to identify disease-associated changes in protein expression within the PSD in bipolar disorder compared with controls. We hypothesized three findings. First, that the altered protein expression would overlap with that observed in schizophrenia PSD.25 Second, that a distinct pattern of altered protein expression would emerge in keeping with genomic and mRNA expression bipolar disorder studies. Third, that mitochondria-associated proteins would be differentially expressed in this PSD-enriched fraction in bipolar disorder .In the current investigations, we enriched for the PSD in the anterior cingulate cortex (ACC) in bipolar disorder and in control human brain samples. This is a candidate brain region in both bipolar disorder and schizophrenia.www.stanleyresearch.org). The series consists of 105 subjects, including 35 schizophrenia, 35 bipolar disorder and 35 control cases. Information on prescribed psychotropic medication is provided by the Stanley Medical Research Institute.Human postmortem brain tissue of the supragenual (BA24) ACC was obtained from the Stanley Medical Research Institute's Array Collection .The enrichment for the PSD was undertaken using methods established previously and recently validated for postmortem brain material.Fifty micrograms of protein from each homogenate was denatured in 10 μl 2% RapiGest solution  at 80°C for 10 min. Samples were subsequently reduced in the presence of 50 mM TCEP (tris2-carboxyethylphosphine)  at 60°C for 60 minutes and alkylated in the dark with 200 mM iodoacetic acid acid (Sigma Aldrich). Protein was digested with 5 μg of sequence grade modified trypsin , in a 37°C shaking incubator for 16 hours. Digestion was stopped and the RapiGest precipitated with formic acid (0.1% v/v). After digestion, peptides were resuspended in 0.5% trifluoroacetic acid, dried in an Eppendorf Vacufuge  and desalted using ZipTips .Label free LC-MS was performed on a Thermo Scientific Q Exactive mass spectrometer connected to a Dionex Ultimate 3000 (RSLCnano) chromatography system. Tryptic peptides were resuspended in 0.1% formic acid. Each sample was loaded onto a fused silica emitter ), packed with Reprocil Pur C18 (1.9 μm) reverse phase media and was separated by an increasing acetonitrile gradient over 120 minutes at a flow rate of 250 nL/min. The mass spectrometer was operated in positive ion mode with a capillary temperature of 320°C, and with a potential of 2300V applied to the frit. All data was acquired with the mass spectrometer operating in automatic data dependent switching mode. A high resolution  MS scan (300–1600 m/z) was performed using the Q Exactive to select the 12 most intense ions prior to MS/MS analysis using HCD.43 The signal for each peptide derived from a given protein is integrated over time (the time it takes to enter the instrument via HPLC), and the summed signals are used to estimate the relative abundance of each protein. To analyze the data, we used the MaxQuant programme specifically for label-free experiments using high resolution instruments supported by Andromeda as a database search engine for peptide identification.44 This program has several statistical control steps to ensure that only high quality reliable ion signals are accepted.We collected two types of mass spectrometry data in the experiments. It is formally a ‘shotgun' experiment and the collected tandem mass spectra were used to identify the proteins. The parent ion signal (MS1 scan) on high resolution instruments such as the Q Exactive used is widely used to calculate protein abundance in so-called ‘label-free' proteomics experiments.45 Carbamidomethylation was defined as a fixed modification, while oxidation and acetylation of the protein N-terminus were defined as variable modifications. Only peptides with seven or more amino acid residues were allowed for identification. Additionally, at least one unique peptide was required to identify a protein. The cut off for false discovery rate for peptide and protein identification was set to 0.01. The label free algorithm takes the maximum number of identified peptides between any two samples and compares the intensity of these peptides to determine peptide ratios.Label free quantitation (LFQ) was performed as previously described.LFQ intensity values were used for protein quantification across the groups.46Raw LFQ intensities were extracted from the MaxQuant software and log base 2 transformed prior to analysis to eliminate distributional skew and to give approximate normality. To avoid bias associated with protein under-representation between groups, proteins were excluded in cases where there was less than 50% availability of the LFQ intensities in each biological group. After data filtering, 45872 LFQ values remained. Regression normalization was performed to remove technical variation between samples.t-test (two-sided) analysis was performed on each protein comparing bipolar disorder with controls.Protein intensities were screened for anomalous and missing data before log transformation (log base 2) and quantile regression normalization. A two-sample 47 of 5% was used to flag those protein identifications deemed statistically significant after adjustment for multiple comparisons. Fold changes were obtained from an exponentiation (power of 2) of the difference in group means. Fold changes <1 were inverted (−1/fold change) to present a consistent scale of measurement.Significance testing was performed at the 5% level, and a false discovery rate (FDR)P-value for the difference between groups was obtained from a nested model  of the corresponding proteins .Family-wise analysis was performed for two families of proteins. Mitochondrial dysfunction and the septin family were investigated as they were highlighted from pathway analysis and visual inspection, respectively. A family-wise The management of data and statistical analyses were carried out with the SAS version 9.2 statistical software  and R version 3.1 statistical software .See 48 namely mitochondrial function (NDUFV2 and SSBP), endocytosis (DNM1 and DTNA) and a member of the Septin family (SEPT11). Validation was undertaken using antibodies to these proteins in the same above-mentioned bipolar disorder and control samples from the Stanley Foundation Array series and animal model of haloperidol-treated rats. For further details on the procedure and specificity of the antibodies used for this study see Proteins were selected for validation based on their degree of differential expression and membership of the main processes and protein families implicated by our current proteomic analysis and comparison with gene expression literature,Thirty-six subjects pooled into 18 samples (8 pairs bipolar disorder and 10 pairs of control) were studied . A totalP⩽0.05). Two hundred eighty-eight proteins were found to be significantly differentially expressed after correcting for multiple testing with FDR<0.05 . The overall fold change for the nine septins in the data set was 1.60, with a 95% confidence interval from 1.34 to 1.92. The 39 mitochondrial dysfunction proteins showed an overall fold change of 2.05 .Furthermore, both the septin and mitochondrial dysfunction set of proteins were differentially expressed in a family-wise analysis , and one in our list of 288 FDR-corrected proteins in the bipolar disorder PSD (FAM162A), and also altered in the same direction. These findings suggest that differential expression of these proteins may be drug-related. However, new western blot analyses for the findings in this study demonstrated that DNM1, DTNA, NDUFV2, SEPT11 and SSBP1 were not significantly altered in rats treated with haloperidol  on the PSD using a rat model of antipsychotic drug treatment.operidol , indicat25 , we found an overlap of 54.5% with the current bipolar PSD differentially expressed data set (78/143), indicating a shared pathophysiology in keeping with past genetic and proteomic49 investigations.We compared the above findings with the previous data obtained from the schizophrenia PSD samplesAs proteins linked to ‘mitochondrial dysfunction' were prominent in the list of differentially expressed proteins in the current study, we asked whether the set of 288 proteins were more likely to have a mitochondrial location in the cell.www.broadinstitute.org/pubs/MitoCarta/)50 we found 97 of the 288 differentially expressed proteins (FDR) as having a mitochondrial localization (Using MitoCarta (lization .P<0.01 Fisher's Exact test). As a group, mitochondrial proteins were generally upregulated  previously shown to be differentially expressed in the PSD in schizophrenia were also differentially expressed in the PSD in bipolar disorder.The findings reported here are relevant to the previous literature, implicating these processes in neuropsychiatric disorders, in particular schizophrenia. Our team recently applied this method to study the PSD in the ACC in schizophrenia and observed altered expression of proteins involved in endocytosis, long-term potentiation and calcium signaling.ling see  were als54 myelination,55 synaptic vescicle fusion,56 membrane curvature57 and cytoskeletal dynamics, which has been implicated in schizophrenia.58 Septin 5, 6 and 11 specifically have been implicated in schizophrenia and bipolar disorder previously in keeping with the proposed synaptic–dendritic basis to the disorders.59Beyond our hypothesized findings, we observed highly significant upregulation for each Septin 3, 5, 7, 8, 9 and 11. Septins have roles in dendritic spine morphology,It is informative to place the differentially expressed bipolar disorder proteome reported here with current genomic literature.14 48 inositol 1,4,5-trisphosphate receptor type 1 , dystrobrevin alpha  and neurotrophic tyrosine kinase receptor.Second, three of our differentially expressed proteins overlapped with the nine genes that were reported in a pathway meta-analysis of bipolar disorder and showed differential expression in the dorsolateral prefrontal cortex in bipolar disorder patients:et al.29 identified 43 genes altered at the mRNA level in the hippocampus in bipolar disorder patients. We have shown that 56% of those were also dysregulated in our differentially expressed bipolar disorder proteome reported here , the degree to which mitochondrial-associated proteins were dysregulated in the current study is more marked than that observed previously in the same Stanley series of brains . This suggests that the mitochondrial-associated protein expression is most marked in the PSD fraction, a finding that may shed light on the findings of several transcriptomic studies, which did not demonstrate prominent alteration in mitochondrial-associated genes.62 Mitochondria are anatomically closely associated with synapses and are essential for the ATP generation, which in turn underpins the protein phosphorylation reactions that mediate synaptic signaling through calcium regulation, exocytosis, vesicle recruitment, potentiation of neurotransmitter release and synaptic plasticity.23 As the provision of an ATP supply power synapse-associated polyribosome complexes and clathrin-dependent endocytic machinery at postsynaptic sites, our observations of altered EIF2 signaling and endocytosis in the PSD in bipolar disorder could potentially be secondary to altered mitochondrial function. EIF2 signaling has been observed recently in two studies of schizophrenia-derived stem cells,64 thus implicating the process of protein translation in both of the major psychosis. The findings raise the testable hypothesis that the major psychoses involve altered mitochondrial function at the level of the synapse that has an impact locally on protein translation and endocytosis, and so on altered synaptic plasticity. Altered trafficking patterns of mitochondria to postsynaptic densities may also contribute, and this possibility is supported by the observed altered expression in the current study of mitochondrial-trafficking proteins KIF1A, DynLL2 and the clathrin mediated endocytosis-associated protein Dynamin1. Further studies are warranted for a deeper understanding of the mitochondrial proteins, and to test whether the mitochondrial changes are indeed primary, and the synaptic changes secondary, as there also is the possibility that this could be the opposite.The most robust finding in the current study is the upregulation of proteins with roles in mitochondrial function. While evidence from many studies implicates mitochondrial function in bipolar disorder, and indeed schizophrenia ), for the current study we used a quadrupole mass filter (Q-Exactive). We confirmed the protein expression changes of a number of proteins that were broadly representative of the main implicated functional pathways and processes using western blotting. We confirmed altered expression of proteins involved in clathrin mediated endocytosis, such as Dynamin1, Dystrobrevin (involved in the formation and stability of synapses), and the mitochondrial proteins NDUFV2 and SSBP; while NDUFV2 is involved in electron transfer, SSBP1 is involved in mitochondrial biogenesis and also confirmed Septin 11.A further strength of our findings is the consistency of the direction of changes of proteins involved in several of the implicated pathways and processes. For example, 38 of the 39 mitochondria-associated proteins as shown by Ingenuity Pathway Analysis to be dysregulated in BPD were increased.65 however, the direction of these influences is inconclusive.67Postmortem studies have limitations that include the well-known confounds such as chronic exposure to neuroleptics, alcohol, tissue pH and postmortem delay that can confound these studies. We designed our study so that age, brain pH and sex were matched closely across groups. We could not account for the potential influence of medication effects, alcohol use, smoking or other recreational substance use, and future work will be needed to address these issues. In particular, it is known that antidepressants and smoking have effect on mitochondrial functions;70 Nanavati et al.69 used proteomic methods to assess the PSD-enriched hippocampus following chronic treatment with lithium or valproate. Overall, there was little overlap with our findings, although they did observe dysregulation of ANK3, IGSF8 and CAPZB in the same direction as observed by us, suggesting that their findings may be drug treatment-related. Two other proteomic studies of the rat prefrontal cortex following chronic lithium or valproate treatment identified CNP, GAP43, MAP2, MAPRE2 and TCEB2 in the same direction as in the bipolar data set, indicating that these findings may be drug treatment-related (see 71 and mitochondrial gene expression27 are reported to be unchanged or largely reduced72 and mood stabilizers are considered to have a neuroprotective effect on mitochondria.30 It should also be appreciated that our understanding of the broader protein content of the PSD is based on proteomic studies of enriched samples and that the methods used can vary, leading to different PSD proteomes. The method used is seen as enrichment rather than a fractionation technique, and there is a possibility that the mitochondrial proteins in the PSD-enriched fraction might come from other cellular locations.In order to account for the potential effect of chronic exposure to neuroleptics in our bipolar disorder cases, we studied the PSD proteome of rats chronically exposed to haloperidol. Few changes were identified following this treatment, although four proteins—FAM163A, RIN1, mTOR and MCCC1—shown to be dysregulated in bipolar disorder were dysregulated in the same direction by haloperidol treatment, suggesting that these effects may have been drug treatment-related see . We alsoated see . HoweverHowever, our PSD enrichment method is well published, and this along with our sensitive LC-MS/MS method yielded a PSD proteome, which is very similar to that described previously.In conclusion, in this study, which is to our knowledge the first of its kind, we have identified and compared the unique set of proteins involved in the PSD in bipolar disorder. Our study provides unique insights into the disorder at the synaptic level and suggests that mechanisms involving altered mitochondrial function, protein translation, calcium signaling, endocytosis and septin function at the level of the PSD are involved in the pathophysiology of bipolar disorder. As these processes act in concert to support synaptic plasticity, they provide insights into the variety of processes that may be targeted in the search for novel treatments for the disorder.


Metabotropic glutamate receptors (mGluRs) are mandatory dimers playing important roles in regulating CNS function. Although assumed to form exclusive homodimers, 16 possible heterodimeric mGluRs have been proposed but their existence in native cells remains elusive. Here, we set up two assays to specifically identify the pharmacological properties of rat mGlu heterodimers composed of mGlu2 and 4 subunits. We used either a heterodimer-specific conformational LRET-based biosensor or a system that guarantees the cell surface targeting of the heterodimer only. We identified mGlu2-4 specific pharmacological fingerprints that were also observed in a neuronal cell line and in lateral perforant path terminals naturally expressing mGlu2 and mGlu4. These results bring strong evidence for the existence of mGlu2-4 heterodimers in native cells. In addition to reporting a general approach to characterize heterodimeric mGluRs, our study opens new avenues to understanding the pathophysiological roles of mGlu heterodimers.DOI:http://dx.doi.org/10.7554/eLife.25233.001 G-protein-coupled receptors (GPCRs) are essential in cell-cell communication and are considered as major drug targets. Although recognized as activating G proteins in a monomeric form , numerouAmong the eight mGluRs, mGlu1 and 5 (group I) are mainly postsynaptic, while mGlu2 and 3 (group II) and mGlu4, 7 and 8 (group III) are predominantly found in presynaptic terminals . HeterolAmong heterodimeric mGluRs, mGlu2-4 was the most studied pair due to its important physiological interest and because different pharmacological tools are available . First, In the present study, we developed two different innovative approaches to characterize the pharmacological and functional properties of mGlu2-4 heterodimers specifically, without any influence of the co-existing mGlu2 and mGlu4 homodimers. We also used an innovative lanthanide-based time-resolved FRET microscopy approach , to demoThe co-expression of both rat mGlu2 and mGlu4 subunits led to the surface expression of three types of dimers: mGlu2 and mGlu4 homodimers and mGlu2-4 heterodimers. We then set up the transfection conditions to obtain an optimal expression at the cell surface of the mGlu2-4 heterodimer . To thatTo examine the effect of various agonists specifically on the mGlu2-4 heterodimer at the surface of live cells, we took advantage of the large conformational change observed at the level of the extracellular domain of mGlu dimers upon activation. This conformational change led to a drastic decrease in TR-FRET signal  that canH = 0.29) on the heterodimer   . In that and C2) , and mGl and C2)  receptor and C2)  (see (Hu and C2)  for the  and C2) . The inh and C2) . The potTaken together, these data revealed a low potency and low Hill coefficient for LY354740, which can be used as a first fingerprint of the mGlu2-4 heterodimer.YADA and mGlu4YADA homodimers, as well as the mGlu2YADA-4YADA heterodimer could not be activated by glutamate (YADA-4 heterodimer (YADA) , which are called YADA mutants , strongllutamate , despitelutamate . Howeverlutamate , consisterodimer . Similarr (YADA) , but mGlr (YADA)  on this H = 0.89 , and theH = 0.89 . These fThe above data prompted us to examine the influence of agonist binding in one subunit on the effect mediated through agonist binding in the second subunit. We observed that agonist binding in mGlu4 receptor increased the potency of mGlu2-specific ligands on the mGlu2-4 heterodimer . In the To quantitatively analyze the effect of mGlu4 agonists on the nH of LY354740 between ligands in the mGlu2-4 heterodimer, mathematical models were developed . AssuminThe synergistic effect between mGlu2 and mGlu4 agonists constitutes a second fingerprint of the mGlu2-4 heterodimer that may be useful for the identification of mGlu2-4 in neurons.20) in the mGlu2 homodimer, but very weakly in the mGlu2-4 heterodimer and not at all in mGlu4  can enhance both agonist affinity and efficacy. They can also have an intrinsic agonist activity on mGluRs . Using oin mGlu4 . Regardiin mGlu4 , as previn mGlu4 . Co-applresponse . Howevertogether . This sytogether . The syntogether  and consIn primary cultures of hippocampal neurons, co-expression of CLIP-mGlu2 and SNAP-mGlu4 subunits could be detected at the cell surface through labeling with CLIP and SNAP substrates carrying either Lumi4-Tb or Red. In these neurons, using a lanthanide-based time-resolved FRET microscope that we recently developed , we deteQ7  and LY354740  induced a significantly large reduction of the fEPSPs in LPP , or by mutating the binding site of either subunit. Accordingly, activating either the mGlu2 or mGlu4 VFT in the mGlu2-4 heterodimer leads to a similar partial effect, both at the conformational level of the VFT dimer, as revealed with the TR-FRET sensor assay, and at the signaling level. This finding is consistent with previous studies demonstrating that two agonists are required, with both VFT closed to fully activate mGlu5 homodimers . HoweverThe effect of LY354740, a well-known group-II-specific mGluR agonist, appears quite complex on the mGlu2-4 heterodimer. This compound has a partial efficacy, but a low Hill coefficient on the heterodimer. Its mGlu2 potency and normal Hill coefficient are restored when the mGlu4 subunit is either activated or mutated to prevent ligand binding. These findings cannot be explained by a simple model in which both mGlu2 and mGlu4 ligands bind selectively to their respective subunit. Instead, our data suggest that LY354740 can bind to both subunits, its interaction with mGlu2 increasing its affinity to mGlu4 in the heterodimer. Such ligand cooperativity between bindings sites in an mGlu dimer has already been reported in mGlu5 receptors in which one binding site is mutated . The synWhen examining the effect of two mGlu2 (BINA and LY487379) and two mGlu4 PAMs (VU0155041 and VU0415374), we confirmed that VU0155041 is the only PAM able to potentiate the effect of glutamate on the mGlu2-4 heterodimer . AlthougA recent study revealed that mGlu2-4 heterodimers are likely present in cortico striatal terminals . This coIt is sometimes argued that GPCR dimers and heterodimers result from the overexpression of the partners. For several reasons, this is unlikely the case for the mGlu2-4 heterodimer. Over-expression is expected to result in larger mGluR complexes since mGluRs are constitutive and covalent dimers , and no Taken together, our data add to previous studies suggesting the existence of mGlu heterodimers in the brain. We show that mGlu2-4 receptors likely exist in the brain and we report innovative approaches that will be useful to confirm the existence of other mGlu heterodimers. For example, one may propose the existence of heterodimers containing an mGlu7 subunit, for which the very low glutamate potency raised a number of questions regarding its roles in vivo. Indeed, mGlu7 can be found with other high-affinity mGluRs, such as mGlu8 , offerinSNAP-Lumi4-Tb, SNAP-Green and CLIP-Green were from Cisbio Bioassays . SNAP-block and CLIP-block were from New England Biolabs . -APDC, ACPT-I, L-AP4, BINA, DCG-IV, LY341495, LY354740, LY487379, VU0155041 and SNC162 were purchased from Tocris Bioscience . LSP4-2022 was kindly provided by Dr. Francine Acher . VU0415374 was synthesized by Dr. Xavier Gómez and provided by Dr. Amadeu Llebaria . Control GFP and ShmRNA mGlu2 and mGlu4 lentiviral particles were purchased from Santa Cruz Biotechnology Inc. . The mGlu4 KO mice were obtained from Dr David Hampson , and thB1), or C2 (the 49-residue coiled-coil region of GABAB2), followed by the endoplasmic reticulum retention signal KKTN, as previously described (NKFTTGSSTNNNEEEKSRLLEKENRELEKIIAEKEERVSELRHLQSRQQLKKTN (the last residues (up to Thr874) of mGlu4 are underlined, those of C1 are in italic). The C-term sequence of CLIP-mGlu2-C2 was previously described  at least 2 hr before the different assays were performed. Frozen-labeled HEK-293 cells were transfected as described above, labeled as described below, then frozen at −80°C with 10% DMSO and fetal bovine serum, and later washed three times in Krebs buffer  0.1%) before use.HEK293 cells  were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% (vol/vol) fetal bovine serum (Sigma Aldrich) in a P2 cell culture room. Absence of mycoplasma was routinely checkedusing the MycoAlert Mycoplasma detection kit , according to the manufacturer protocol. HEK 293 cells were used after 35 to 40 passages and transfected with a reverse transfection protocol using Lipofectamine 2000 , and finally plated in polyornithine-coated, black-walled, dark-bottom, 96-well plates at 10In order to optimize the best expression of mGlu2-mGlu4 heteromers, several ratios of mGlu2:mGlu4 were assayed. It was determined by TR-FRET analysis that 2:1 ratio (40 ng CLIP-mGlu2: 20 ng SNAP-mGlu4) was optimal for the detection of all populations . Using tQ7 striatal neuronal progenitor cell line . The emission decay was collected during 2500 or 5000 μs with 5 μs or 10 μs steps, respectively, at 620 nm for the donor (Lumi4-Tb) and at 520 nm for Green, as can be indicated in the ‘advanced mode’ option of the plate-reader’s software. For acceptor ratio determination, optimal integration windows were determined as previously reported . HEK293 cells were co-transfected with the indicated mGluR plasmids and the pGloSensor-22F plasmid. The day after, cells were starved during 2 hr in serum-free medium and afterwards incubated in Krebs buffer with 450 μg/ml luciferin (Sigma-Aldrich) during 1 hr. The luminescence peak signal was measured on a Mithras microplate reader at 28°C during 8 min since luminescence signal was stable. Then, forskolin plus mGluR ligands were added and luminescence was measured for 30 min.The amount of cAMP was determined using the Glosensor4 cells/well and introduced into the incubator at 33°C overnight. Medium was replaced by serum-free medium during 2 hr to reach a stable cell index, and then forskolin and mGluR ligands were added and the signal was followed during at least 2 hr using xCELLigence RTCA DP apparatus . When antagonists or PAMs were used, they were added 20 min before forskolin. Pertussin toxin was added 4 hr after cell plating and incubated overnight.xCELLigence plates were coated with poly-ornithine and laminin during 1 hr, and neuronal cells were seeded at 3 × 102. Neurons were cultured in Neurobasal medium (Thermo Fisher Scientific) supplemented with 2% B-27 (Thermo Fisher Scientific), 100 U/ml Penicillin-Streptomycin (Thermo Fisher Scientific), 10 mM HEPES, and 0.5 mM Glutamax medium (Thermo Fisher Scientific). 0.5 mM L-glutamine was added when plating the cells. Half of the medium was exchanged weekly. Neurons were transfected with Lipofectamine 2000 at 10 days in vitro (DIV). The medium was exchanged after 4 hr of incubation with the transfection reagent with half fresh medium and half medium conditioned by incubation with primary neurons. pRK5 plasmids for expression of SNAP- or CLIP-tagged rat mGluRs under control of the CMV promoter were previously described  supplemented with 1% BSA followed by a wash in imaging buffer with 1% BSA and three washes in imaging buffer. Cells were imaged in imaging buffer. Images were acquired with a homebuilt TR-FRET microscope (0.5) was done with the PixFRET plugin to ImageJ (Hippocampi from Sprague-Dawley rat embryos on embryonic day 18 (E18) were dissected, dissociated by treatment with liberase TL , then mechanical triturated and plated on Lab-Tek II chambered cover slides (Thermo Fisher Scientific) coated with poly-L-ornithine and laminin (Sigma-Aldrich) at a density of ~300 neurons/mmescribed . For inccroscope . Brieflycroscope . Correcto ImageJ . Accepto2PO4, 25 mM NaHCO3, 1 mM MgCl2, 2 mM CaCl2, and 10 mM glucose, pH 7.4, equilibrated with 95% O2 and 5% CO2. Parasagittal acute 400-µm-thick slices were prepared with a vibratome  in ice-cold ACSF. Sections were kept at room temperature for at least 1 hr before recording. Slices were transferred to a submersion recording chamber, maintained at 30°C and perfused with oxygenated ACSF at a rate of one chamber volume (1.5 ml) per minute.Acute slices were prepared from adult (P21-P30) control or mGlu4-KO mice following a protocol approved by the European Communities Council Directive and the French low for care and use of experimental animals. Mice were decapitated, and brains quickly removed and chilled in cold artificial cerebro-spinal fluid (ACSF) containing 125 mM NaCl, 2.5 mM KCl, 1.25 mM NaHfEPSPs were evoked at 0.033 Hz using bipolar stimuling electrode and recorded using glass micropipettes (3–5 MΩ) and filled with 3 M NaCl. Stimuling electrode was placed in the outer thirds of the molecular layer of the dentate gyrus for stimulation of the lateral perforant path (LPP). Correct positioning of electrodes was verified by application of paired-pulse at an interval of 100 ms induces paired-pulse facilitation in the LPP. The effect of paired-pulse stimulation was assessed and only those slices that displayed the correct facilitation in the LPP were used for this study. Input-output curves were generated for each slice, and the stimulation intensity was adjusted to 70% of the maximum response. Baseline fEPSPs were recorded for a minimum of 20 min before bath-application of different agonists or PAMs. Evoked responses were analyzed by measuring the slope of individual fEPSPs. The slopes from two sequential sweeps were averaged. All slopes were normalized to the average slope calculated during the pre-drug period (percentage of baseline). All data were analyzed offline using pClamp 9 (Molecular Devices) and are reported as the mean ± SEM. Statistical comparisons were made using two-tailed unpaired or paired Student’s t-tests. Differences were considered significant at p<0.05. The % inhibition was calculated by the difference of the slope between the baseline and the last 4 min of the drug application.SAS/STAT 9.4  statistical package was used for parameter optimization and statistical analyses in mathematical modeling. Curve fitting was performed using nonlinear regression using GraphPad Prism software. p-values<0.05 were considered statistically significant using one-way ANOVA with Bonferroni post-hoc test. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Richard Aldrich as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Robert Duviosin (Reviewer #3).Thank you for submitting your article "Pharmacological evidence for a metabotropic glutamate receptor heterodimer in neurons" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.The manuscript was evaluated by knowledgeable referees, who provided positive comments about the findings of a mGluR2-4 heterodimer. Using a time resolved FRET based approach, strong evidence was provided for a heterodimeric arrangement of mGlu2-4 in HEK293 cells and primary neurons after transfection experiments. Agonists of mGlu2 were used to define the responses and cooperativity of the Glu2-4 heterodimer with a determination of the Hill coefficient. Several reservations were raised, including (1) the relevance of the findings to the endogenous receptor; (2) the need for additional data in neuronal systems where mGlu2-4 receptors are found; and (3) a number experimental details need to be clarified and rectified throughout the manuscript. It was felt that the Background and Materials and methods require more explanation, since the methods represent a new way of demonstrating heteromer interactions. We feel that the majority of these issues can be addressable in a timely fashion and recommend a major revision for this paper. The major concerns are listed below.Review #1:Although the analysis is compelling, the study would be strengthened if the transfection experiments excluded the effects of overexpression and miss localization of the receptors.1) An expression system is used to follow the mGlu2-4 heterodimers at the cell surface, but this does not exclude an intracellular localization of the receptors. Also, strong promoters were used to gain high expression levels in HEK293 and primary hippocampal neurons. Some attention should be given to appropriate cellular expression after transfection. The images in 2) A SThdHQ7 striatal cell line is used to follow mGlu2-4 receptors. Why do the cells shrink after activation of adenylate cyclase (Results section)? There is not a good explanation for why these cells are chosen. What is the relevance of mGlu2-4 to striatal disorders, such as Huntington's disease?”) would be helpful.3) How is "minimum leaking of respective homodimers" determined? A definition of the "orthosteric binding site" of mGluR (subsection “Activation of both subunits in mGlu2-4 receptor is required for full activity4) The stoichiometry of many other receptor systems has been deduced from affinity crosslinking experiments. Given all the tools available to the laboratory, this is an accessible experiment and would provide more evidence for the heterodimer model.Reviewer #2:In this manuscript the authors examine heterodimerization between metabotropic glutamate receptor types in modulating receptor activity. The majority of the studies are carried out with recombinant receptors- while the studies are solid and address an important question, a cursory effort has been made to characterize the native receptors. The authors use primarily heterologous or neuronal cell lines and FRET-based techniques to explore the changes in movement of the receptors upon ligand binding. They go on to explore the regulation of signaling altered by this process. The studies are well thought out and executed and conclusions are based on solid data. In sum, the majority of the studies are carried out with recombinant receptors expressed in heterologous cells or cultured neurons – while the studies are solid and address an important question in the field, how relevant this information is to the native receptor in an endogenous setting is not well explored.1) Title – as written the title makes a sweeping claim that is only partially addressed and so is misleading.2) Abstract – This is written in rather general terms and reads like conclusions rather than the synopsis of the study. Expanding the Abstract to include a bit more information as to the major findings of the study would help.3) Materials and methods – More details need to be included in this section and some important details are missing; for example, details of the receptors (including species) transfected into neurons is not included here . How the ER-retaining receptors were generated, characterized and independently tested so that only heteromers appear at the cell surface is not clear. Authors are encouraged to show this data as it would strengthen their argument that only heteromers are expressed at the cell surface. These are only a couple of examples and the authors are should carefully go over each of the experiments in the manuscript to ensure that all of the methods are clearly described rather than having the reader go back and forth between the Materials and methods and the figure legend section.Results”: As written it appears that the concentrations of SNAP-donor and CLIP-acceptor substrates that gave a signal for the heteromer did not give a TR-FRET signal with respective receptor homomers – is this true? This needs to be clarified in the manuscript.1)Subsection “A FRET-based sensor to identify mGlu2-4 specific fingerprints2). In the same subsection, paragraph two: 3)A table with potencies, efficacies, Hill coefficient with the different ligands in cells expressing homomers and heteromers would be useful as it would enable the reader to compare changes instead of going through different figures to check out statements made in the text.4)5) Also a dose response curve of either LY34540 (or LSP4-2022) in the presence of a low dose of LSP4-2022 (or LY34540) would have strengthened this crucial point.6) 7.)There is a mismatch between the title of the figure and figure legend – the authors are requested to check this carefully. For example, legend on 8) Most importantly, neither the co-localization of the two receptors in this system nor the absence of synergy in animals lacking one of the receptors has been explored.Reviewer #3:This manuscript presents very nice data in support of mGlu2-mGlu4 heterodimer receptors. The FRET experiments are convincing and demonstrate that the pharmacology of herodimers can be distinguished from that of either homodimers. Second, the authors show that several compounds have distinct actions on hererodimers compared to homodimers, such as different Hill coefficients or partial agonist or synergistic activities. Finally the experiments measuring cell swelling of cultured striatal neuron cell line as a proxy for adenylate cyclase activation are inovative and reveal the synergistic activity of BINA and VU0415374 in neurons. My only question about this manuscript is that the evidence of mGlu2-4 heterodimers in the lateral perforant path is most suggestive, but it is not conclusive. The synergistic effects shown could be caused downstream of the receptors in the Gi-coupled pathways. Given the pharmacological fingerprints presented in  The manuscript was evaluated by knowledgeable referees, who provided positive comments about the findings of a mGluR2-4 heterodimer. Using a time resolved FRET based approach, strong evidence was provided for a heterodimeric arrangement of mGlu2-4 in HEK293 cells and primary neurons after transfection experiments. Agonists of mGlu2 were used to define the responses and cooperativity of the Glu2-4 heterodimer with a determination of the Hill coefficient. Several reservations were raised, including;1) The relevance of the findings to the endogenous receptor;Firstly, our data indicate that the formation of mGlu2-4 heterodimers is not the consequence of an over-expression of these subunits. Of note, the expression levels of both mGlu2 and mGlu4 are rather low in the striatal cell line, such that no direct responses could be measured using conventional second messenger assays. This is only when using a label free assay that mGluR responses could be measured.Also, our quantification of transfected mGlu2 receptors in hippocampal neurons revealed a 5-fold only increase in cell surface receptors compared to controls.We also report that the synergistic effect observed in hippocampal slices is lost in the mGlu4 KO mice, and that the increase in potency of the mGlu2 agonist LY354740 observed upon activation of the mGlu4 subunit cannot be reproduced by activating a co-expressed the Gi-coupled δ opioid receptor. We think our study brings strong pharmacological evidence for the existence of mGlu2-4 heterodimers in cells naturally expressing these receptors, and that this phenomenon is not the consequence of receptor over-expression. We agree that more work is needed to decipher the physiological significance of such heteromeric assemblies, but this is not the scope of our present manuscript that mainly concentrated on the development of assays aimed at characterizing mGlu heterodimers.2) The need for additional data in neuronal systems where mGlu2-4 receptors are found;Data with mGlu4 KO mice have been added, and confirmed that the LSP4-2022 effect is mediated through the mGlu4 subunit. As expected, no synergistic effect is observed between LY354740 and LSP4-2022 in the absence of mGlu4.3) A number experimental details need to be clarified and rectified throughout the manuscript. It was felt that the Background and Materials and methods require more explanation, since the methods represent a new way of demonstrating heteromer interactions. We feel that the majority of these issues can be addressable in a timely fashion and recommend a major revision for this paper. The major concerns are listed below.The Materials and methods section has been completed with as much information as possible to allow any reader to repeat the experiments.Review #1:Although the analysis is compelling, the study would be strengthened if the transfection experiments excluded the effects of overexpression and miss localization of the receptors.1) An expression system is used to follow the mGlu2-4 heterodimers at the cell surface, but this does not exclude an intracellular localization of the receptors. Also, strong promoters were used to gain high expression levels in HEK293 and primary hippocampal neurons. Some attention should be given to appropriate cellular expression after transfection. The images in There are many questions raised in this first point of the referee that we will clarify, one by one.Regarding the validity of the quality control system used to allow a specific cell surface expression of the mGlu2-4 heterodimers, important information can be found in The referee is right that either assay  are conducted in the presence of intracellular receptors. However, when using the biosensor, because we used non cell permeant CLIP and SNAP substrates, only cell surface receptors carrying an extracellular CLIP or SNAP tags can be labeled. Only those cell surface receptors will then be recorded in any biosensor assays. Regarding the ER retention of the C1 and C2 constructs, although retained intracellularly, and even though some authors argue that intracellular mGluRs can signal, we have never been able to measure any signaling with the C1 or C2 constructs expressed alone upon glutamate application . We modified the text to make this point clearer to the readers.The referee also questions the possibility that the reported data results from an over-expression of the receptors. We don't think this is the case, for the following reasons.i) The interaction between cell surface receptors may be artificially favored due to an over-expression of the proteins. This is well illustrated by the work by Calebiro et al., 2013 reporting that the proportion of dimers and oligomers increases with receptor density at the cell surface. Under our experimental condition, although we can measure TR-FRET between subunits, we have not been able to measure significant signal under conditions where TR-FRET can only result from the proximity between dimers . Moreover, various experimental protocols were used to estimate the size of the mGlu2-4 complexes and all were consistent with strict heterodimers .ii) The functional data obtained with the striatal cell line  nicely ithat a receptor with the pharmacological properties of the mGlu2-4 heterodimer can be functionally detected whereas these cells express a low amount of each receptor as evidence by the difficulty in measuring a second messenger response upon agonist activation. Of note, these functional data were obtained in control nontransfected cells naturally expressing mGlu2 and mGlu4.iii) The mGlu2 receptor expression in transfected neurons has been quantified, and is reported in another study to be submitted soon. Because a few percent of the cultured neurons are transfected under our experimental conditions, radioactive ligands could not be used to quantify mGlu2 receptor expression. We then took advantage of an mGlu2 specific nanobody (publication submitted) labeled with a fluorophore to compare the fluorescent signal in non-transfected neurons and in transfected neurons. Data are shown in the A specific paragraph in the Discussion has been added to discuss this issue.The last aspect raised in this first point of the referee relates to the subcellular localization of the receptors. As illustrated in Data shown in 2) A SThdHQ7 striatal cell line is used to follow mGlu2-4 receptors. Why do the cells shrink after activation of adenylate cyclase (Results section)? There is not a good explanation for why these cells are chosen. What is the relevance of mGlu2-4 to striatal disorders, such as Huntington's disease?Forskolin induces the shrinking of several cell lines. Indeed, forskolin is one of the classical components of differentiation cocktails  36, 52–56; BMC Cell Biol. 2010 Apr 16;11:25). STHdh, can be differentiated using a cocktail containing forskolin. During our initial studies with these cells, and looking for mGlu2 or mGlu4 inhibition of forskolin-induced cAMP production, we came up to this interesting observation that can be easily followed using the Xcelligence label-free system. We then used this characteristic to evaluate Gi protein activation by mGlu2 and mGlu4 agonists.STHdh cells are well characterized and largely used as a neuronal model. These cells express different key proteins expressed in striatal neurons . As a search for a neuronal cell line to test the possible formation of mGlu24 heterodimers, we chose these cells as an alternative to cultured neurons as such cells are easier to transfect. Of note, mGlu4 is known to be expressed in some striatal neurons  . Although mGlu2 receptors are found in the striatum , these mostly correspond to pre-synaptic receptors in cortico-striatal terminals  since low in situ hybridization signal could be detected in striatal cells .We then checked whether mGlu2 and or mGlu4 mediated responses could be detected in these cells, and it was the case using the sensitive label free assay.These cells were primarily chosen as a neuronal cell model, rather than a model for striatal neurons with the possibility to propose hypothesis regarding the role of mGlu2-4 heterodimer in the basal ganglia network, and their possible use to treat Huntington or Parkinson diseases. We think a more integrated work with more native situation will be necessary before entering into such discussion.”) would be helpful.3) How is "minimum leaking of respective homodimers" determined? A definition of the "orthosteric binding site" of mGluR . To clarify the issue, we replaced “orthosteric” by “glutamate” so that non-specialist readers will understand that we mutated the binding site where the natural ligand binds.4) The stoichiometry of many other receptor systems has been deduced from affinity crosslinking experiments. Given all the tools available to the laboratory, this is an accessible experiment and would provide more evidence for the heterodimer model.Although we agree that crosslinking experiments can be informative, such experiments are also highly criticized because transient association can be sufficient for crosslinking. Accordingly crosslinked species will accumulate during the duration of the crosslinking experiment. A nice example is reported in our recent paper  where crosslinking could be observed between mGlu2 dimers providing these are in the active form. Such crosslinking resulted in the appearance of FRET between dimers. However, no such FRET could be detected without crosslinking, indicating that only a very small proportion of the dimers were associated into larger complexes under control condition, and that such complexes accumulated upon crosslinking.To us, the best illustration that mGlu2 and mGlu4 associate into heterodimers, rather than the association between two homodimers, is the large change in FRET upon receptor activation, illustrating that the FRET really originates from a specific association of the subunits as observed with the homodimers. In addition, in our previous study  we used several approaches to demonstrate that mGlu2 and mGlu4 form heterodimers at the cell surface rather than larger complexes. We now refer to this previous study in our revised manuscript.Reviewer #2:In this manuscript the authors examine heterodimerization between metabotropic glutamate receptor types in modulating receptor activity. The majority of the studies are carried out with recombinant receptors- while the studies are solid and address an important question, a cursory effort has been made to characterize the native receptors. The authors use primarily heterologous or neuronal cell lines and FRET-based techniques to explore the changes in movement of the receptors upon ligand binding. They go on to explore the regulation of signaling altered by this process. The studies are well thought out and executed and conclusions are based on solid data. In sum, the majority of the studies are carried out with recombinant receptors expressed in heterologous cells or cultured neurons – while the studies are solid and address an important question in the field, how relevant this information is to the native receptor in an endogenous setting is not well explored.1) Title – as written the title makes a sweeping claim that is only partially addressed and so is misleading.The title has been modified as follow: "Pharmacological evidence for native metabotropic glutamate receptor heterodimers in neuronal cells"We still think the data obtained with the neuronal cell line, support the existence of native heterodimeric mGlu2-4 in non-transfected cells. Although our data in hippocampal slices are not fully conclusive, as discussed with referee #3, still they are consistent with our proposal. Indeed, such a strong synergy between Gi-coupled receptor agonists has never been observed through signaling synergism. Because our study was primarily aimed at establishing innovative approaches to specifically study the properties of mGlu heterodimers, and because the identified properties can be observed in cells naturally expressing both mGlu2 and mGlu4, we think our new proposed title is appropriate.2) Abstract – This is written in rather general terms and reads like conclusions rather than the synopsis of the study. Expanding the Abstract to include a bit more information as to the major findings of the study would help.We still think the information given in the Abstract is necessary for a wide readership. Unfortunately, the Abstract could not be extended due to length constraint.3) Materials and methods – More details need to be included in this section and some important details are missing; for example, details of the receptors (including species) transfected into neurons is not included here . How the ER-retaining receptors were generated, characterized and independently tested so that only heteromers appear at the cell surface is not clear. Authors are encouraged to show this data as it would strengthen their argument that only heteromers are expressed at the cell surface. These are only a couple of examples and the authors are should carefully go over each of the experiments in the manuscript to ensure that all of the methods are clearly described rather than having the reader go back and forth between the Materials and methods and the figure legend section.We are really sorry for not checking carefully the Materials and methods section before submission of our manuscript. We thank the referee for pointing all these missing parts. The Materials and methods section has now been complemented and includes all details the reviewer requested. Controls using ER-retaining constructs can be found in Results”: As written it appears that the concentrations of SNAP-donor and CLIP-acceptor substrates that gave a signal for the heteromer did not give a TR-FRET signal with respective receptor homomers – is this true? This needs to be clarified in the manuscript.1) Subsection “A FRET-based sensor to identify mGlu2-4 specific fingerprintsWhen using as substrates SNAP-donor and CLIP-acceptor, then the homodimers composed of two SNAP-tagged subunits only carries a donor, such that no FRET signal is generated. Similarly, the CLIP-tagged homodimer is labeled with the acceptor only, and will also not generate a FRET signal. Then, as indicated in the text, only the heterodimer composed of one subunit tagged with a SNAP, and another subunit tagged with a CLIP, will be labeled with both a donor and an acceptor, such that TR-FRET signals will be generated by the heterodimer only. This doesn’t mean (as suggested by the referee’s comment) that homomers cannot be formed, but simply that our experimental conditions allow to monitor by FRET signals coming from the heteromers, exclusively.This point has been made clearer in our revised manuscript.2). In the same subsection, paragraph two: This has been corrected.3)A table with potencies, efficacies, Hill coefficient with the different ligands in cells expressing homomers and heteromers would be useful as it would enable the reader to compare changes instead of going through different figures to check out statements made in the text.The requested Table has been added .4)The requested information has been added in the figure and figure legend.5) Also a dose response curve of either LY34540 (or LSP4-2022) in the presence of a low dose of LSP4-2022 (or LY34540) would have strengthened this crucial point.It is difficult to perform a dose response curve in a slice preparation. Instead of doing these experiments, we preferred to generate data from slices prepared from mGlu4 KO mice (see revised 6) The colors are related to the FRET intensity . We want to make clear that the three graphs were generated from the same set of cells transfected with various amount of CT-mGlu2 and SNAP-mGlu4. The TOP graph reveals FRET data after CLIP labeling with both donor and acceptor substrates. It shows the best conditions leading to high amount of mGlu2 homodimers. The middle graph reports data after labeling the cells with donor and acceptor SNAP substrates. It shows the best conditions to get higher amount of mGlu4 homodimers. The bottom graph shows data after labeling the cells with a SNAP donor substrate, and a CLIP acceptor substrate. Then this bottom graph shows the best condition to get higher proportion of mGlu2-4 heterodimers. By comparing the three graphs, one can identify the conditions that favor the heteromer expression relative to each of the homodimers. The figure legend has been modified.7.)There is a mismatch between the title of the figure and figure legend – the authors are requested to check this carefully. For example, legend onsays mGlu4 but title of figure says mGlu2, legend forsays mGlu2 but title of figure says mGlu4. Similarly intitle in the figure says mGlu2 agonist but Results section states mGlu4 agonist.We thank the referee for pointing these mistakes. These have been corrected.8) Most importantly, neither the co-localization of the two receptors in this system nor the absence of synergy in animals lacking one of the receptors has been explored.Experiments have been performed in slices prepared from mGlu4 KO mice. The data are now included in the revised Regarding the co-localization studies, it is known that both mGlu2 and mGlu4 are expressed in these terminals, and our data confirm this since both LY354740 and LSP4-2022 inhibit transmission. Performing co-localization studies would need more time for us to conduct such studies especially because these would need electron microscopy to really be meaningful. We thought that such studies are out of the scope of the present study, concentrating in original approaches to specifically study the pharmacological properties of mGlu heterodimers. We also preferred to concentrate on the time available to produce a revised manuscript, on the characterization of mGlu4 KO slices.Reviewer #3:This manuscript presents very nice data in support of mGlu2-mGlu4 heterodimer receptors. The FRET experiments are convincing and demonstrate that the pharmacology of herodimers can be distinguished from that of either homodimers. Second, the authors show that several compounds have distinct actions on hererodimers compared to homodimers, such as different Hill coefficients or partial agonist or synergistic activities. Finally the experiments measuring cell swelling of cultured striatal neuron cell line as a proxy for adenylate cyclase activation are inovative and reveal the synergistic activity of BINA and VU0415374 in neurons. My only question about this manuscript is that the evidence of mGlu2-4 heterodimers in the lateral perforant path is most suggestive, but it is not conclusive. The synergistic effects shown could be caused downstream of the receptors in the Gi-coupled pathways. Given the pharmacological fingerprints presented in The referee is right, one cannot exclude the possibility of a synergistic effect between two Gi coupled receptors, although such a synergy has not been reported so far. We therefore modified our manuscript to minimize our conclusion of the existence of mGlu2-4 heterodimers in the perforant path terminals, and rather claim that our data are consistent with the presence of such heterodimers. In order to offer a stronger conclusion, we aimed at studying, in recombinant systems, the possible synergistic effects between mGlu2 and mGlu4 homodimers. Although no such synergy could be detected with the TR-FRET biosensors , and examined whether activation of DOR could potentiate the effect of the mGlu2 agonist in inhibiting Forskoline-induced cAMP formation using the pGlo-sensor. As show in Although not fully conclusive, we think these additional data strengthen our view.Regarding the last comments of the referee, though we agree that such experiments would be great, it is quite difficult, time consuming, and expensive in terms of the amount of drug needed to perform a full dose-response curve using electrophysiological recordings in brain slices. We then preferred to concentrate our work in getting data with the mGlu4 KO mice.