Cadherins contribute to the organization of nearly all tissues, but the functions of several evolutionarily conserved cadherins, including those of calsyntenins, remain enigmatic. Puzzlingly, two distinct, non-overlapping functions for calsyntenins were proposed: As postsynaptic neurexin ligands in synapse formation, or as presynaptic kinesin adaptors in vesicular transport. Here, we show that, surprisingly, acute CRISPR-mediated deletion of calsyntenin-3 in mouse cerebellum in vivo causes a large decrease in inhibitory synapse, but a robust increase in excitatory parallel-fiber synapses in Purkinje cells. As a result, inhibitory synaptic transmission was suppressed, whereas parallel-fiber synaptic transmission was enhanced in Purkinje cells by the calsyntenin-3 deletion. No changes in the dendritic architecture of Purkinje cells or in climbing-fiber synapses were detected. Sparse selective deletion of calsyntenin-3 only in Purkinje cells recapitulated the synaptic phenotype, indicating that calsyntenin-3 acts by a cell-autonomous postsynaptic mechanism in cerebellum. Thus, by inhibiting formation of excitatory parallel-fiber synapses and promoting formation of inhibitory synapses in the same neuron, calsyntenin-3 functions as a postsynaptic adhesion molecule that regulates the excitatory/inhibitory balance in Purkinje cells. Synapses mediate information transfer between neurons in brain and process the information during transfer. In processing information, synapses are dynamic: Synapses are continuously eliminated and newly formed throughout life, and are additionally restructured by various forms of synaptic plasticity during their life cycle . SynapseClstn1-3 in mice) and a single gene in Drosophila, C. elegans, and other invertebrates  are atypical cadherins that are encoded by three genes in mammals  of APP and of Aβ peptides without changing APP levels, making it difficult to understand how a decreased APP cleavage causing increased CTF levels could also elevate Aβ levels , whereas other cerebellar neurons do not   , suggestr cortex , the motClstn3 KO impaired motor behaviors by a synaptic mechanism as suggested by current views on calsyntenin functions (see Introduction), we first examined inhibitory synapses on Purkinje cells, which are the only postsynaptic targets for all excitatory and inhibitory inputs in the cerebellar cortex. We quantified the inhibitory synapse density in the cerebellar cortex using immunohistochemistry for vGAT and GABA-Aα1 receptors, which are pre and postsynaptic markers for inhibitory synapses in Purkinje cells  was observed in the deep molecular layer , without changing the mIPSC amplitude  from Purkinje cells in the presence of tetrodotoxin . The Clsmplitude . Moreovef mIPSCs . Measureoperties .Clstn3 deletion, we analyzed mIPSCs as function of their amplitudes, with the notion that more distance inhibitory synapses derived from stellate cells would have lower amplitudes. First, we plotted the mIPSC amplitudes in a normal distribution  in the vGluT1 staining intensity of both the superficial and the deep molecular layers of the cerebellar cortex  in GluA2 staining in Purkinje cells than that observed for vGluT1 staining  in synaptic GluA1 staining intensity  in the density of spines in the superficial area of the cerebellar cortex, and a trend toward an increase in the deep area of the cerebellar cortex  in the presence of tetrodotoxin. We observed an increase in mEPSC amplitudes (~25%) and frequency (~15%) in kinetics . The majkinetics . To prefkinetics . The resactivity .Clstn3 KO robustly enhanced parallel-fiber synaptic responses (~60% increase)  . This fi altered . Thus, tClstn3 KO-induced alteration. Specifically, the amplitude, paired-pulse ratio, and kinetics of climbing-fiber EPSCs in control and Clstn3 KO Purkinje cells were indistinguishable , the adhesion molecule acts to promote synaptic function in both , but also that Clstn3 increases the numbers of another specific synapse (GABAergic basket- and stellate-cell synapses) in the same neurons.The functions we describe here for  in both . Not onlClstn3 at much higher levels than Clstn1 or Clstn2, whereas other cerebellar neurons predominantly express Clstn1 and exhibit only low levels of Clstn3  may regulate the loudness of different sections of the orchestra, or translated into the terms of a synapse, control the efficacy of signals regulating excitatory vs. inhibitory synapses. However, it is also possible that Clstn3 acts directly in the process of synapse formation itself, but differentially affects distinct synapses depending on the presence of different ligands – time will tell!Multiple synaptic adhesion molecules have already been implicated in synapse formation in Purkinje cells. The interaction of presynaptic neurexins with cerebellins and postsynaptic GluD receptors plays a major role in shaping parallel-fiber synapses , and theje cells , as is dje cells . How do https://www.jax.org/strain/024858) were used and maintained as homozygotes (Constitutive Cas9 mice (ozygotes . Pcp2-Crhttps://zlab.bio/guide-design-resources) to minimize potential off-target effects. The pAAV construct was modified from Addgene #60231 (https://tide.nki.nl/) (Single-molecule RNA fluorescent in situ hybridization (smRNA-FISH) smRNA-FISH in-situ hybridization experiment was performed on brain sections from P30 wild type C57BL/6 J mice according to the manufacturer instructions using Multiplex Fluorescent Detection Reagents V2 kit . Predesigned probes for Clstn1 (# 542611), Clstn2 (# 542621), and Clstn3 (# 542631) were purchased from ACD. sgRNA design and generation of Vectors sgRNAs were designed using protocols developed by the Zhang lab (e #60231  with Cree #60231 . Brieflye #60231 . PCR pronki.nl/) .Primers for sg66 and sg21 editing site sequencing:Primers for off-target site sequencing on sg66:Primers for off-target site sequencing on sg21:2 culture area, using the calcium phosphate method. Cells were harvested 72 hr post-transfection, and nuclei were lysed and AAVs were extracted using a discontinuous iodixanol gradient media at 65,000 rpm for 3 hr. AAVs were then washed and dialyzed in DMEM and stored at –80 °C until use. Genomic titer was tested with qPCR and adjusted to 5 × 1012 particles/ml for in vivo injections.AAV: pAAV carrying sgRNAs was serotyped with the AAV-DJ capsid . Briefly2 dish area, respectively. Media with viruses was collected at 36–48 hr after transfection, centrifuged at 3000 x g for 20 min to remove debris, filtered at 0.45 μm, and ultracentrifuged at 55,000 x g for 2 hr. Pellets were re-suspended with 300 μl DMEM for every 30 ml of the initial volume to achieve a lower titer. Viruses were aliquoted and stored at –80 °C until further use.Lentivirus: U6-sgRNAs or controls, and CAG-tdTomato were cloned into the lentiviral backbone FUW and lentiviruses were produced with three helper plasmids  in HEKT293 cells (ATCC) using calcium phosphate, at 5 μg of each plasmid per 25 cmStereotactic injection: p21 Cas9 mice were anesthetized with tribromoethanol , head-fixed with a stereotaxic device (KOPF model 1900). AAVs carrying sgRNAs or control viruses were loaded via a glass pipette connected with a 10 μl Hamilton syringe  on a syringe injection pump  and injected at a speed of 0.15 μl/min. Pipette was left in cerebellum for additional 5 min after injection completion. Carprofen (5 mg/kg) was injected subcutaneously as anti-analgesic treatment. To infect the whole cerebellum, we injected multiple sites evenly distributing over the cerebellum skull, coordinates were as previously reported , anterioClstn3 mRNA level. To detect genome editing efficiency, qPCR primers and probe were targeting the two exons and designed to flank the double-strand breaks of the two sgRNAs .Virus-infected cerebellar tissue indicated by tdTomato was carefully dissected under fluorescence microscope. RNA was extracted using Qiagen RNeasy Plus Mini Kit with the manufacturer’s protocol . Quantitative RT-PCR was run in QuantStudio 3  using TaqMan Fast Virus 1-Step Master Mix . PrimerTime primers and FAM-dye coupled detection probes were used for detecting o sgRNAs . Rα1 subunit antibody staining, antigen retrieval was achieved via pepsin digestion according to antibody instruction and as previously described . Signals were detected with Odyssey CLx imaging systems (LI-COR) and data were analyzed with Image Studio 5.2 software. Total intensity values were normalized to actin prior to control.2, 0.1 CaCl2, 25 glucose, 1.25 NaH2PO4, 0.4 ascorbic acid, 3 myo-inositol, 2 Na-pyruvate, and 25 NaHCO3). 250 μm sagittal slices were cut using a vibratome  and recovered at room temperature for ＞1 hr before recording. Oxygenated ACSF  was perfused at 1 ml/min during recording. Whole cell recordings with Purkinje cells were from cerebellar lobules IV/V, with patch pipettes (2–3 MΩ) pulled from borosilicate pipettes  using PC-10 puller . The following internal solutions were used (in mM): (1) for EPSC, 140 CsMeSO3, 8 CsCl, 10 HEPES, 0.25 EGTA, 2 Mg-ATP, 0.2 Na-GTP (pH adjusted to 7.25 with CsOH); (2) for IPSC, 145 CsCl, 10 HEPES, 2 MgCl2, 0.5 EGTA, 2 Mg-ATP, 0.2 Na-GTP (pH adjusted to 7.25 with CsOH). Liquid junction was not corrected during all recordings. For all EPSC recordings, 50 μM picrotoxin  and 10 μM APV  were contained in ACSF, and (1) additionally 0.5 μM NBQX  were included for climbing-fiber EPSC recordings; (2) 1 μM TTX  for mEPSC recordings. For all IPSC recordings, (1) 10 μM CNQX  and 10 μM APV were included in ACSF, and (2) 1 μM TTX were included in mIPSC recordings. A glass theta electrode  filled with ACSF was used as stimulating electrode, and littermate control and Clstn3 knockout mice were analyzed meanwhile using the same stimulating electrode. For climbing fibers, the electrode was placed in the granule cell layer around Purkinje cells and identified by all-or-none response, together with paired-pulse depression at 50ms inter-stimulus interval  and analyzed with Clampfit10.4. Coefficient of variation was calculated according to previous report .Two mg/ml Biocytin  was dissolved in Cs-methanesulfonate internal solution followed by whole-cell voltage clamp recordings in the Purkinje cells . Slices Clstn1, Clstn2, Clstn3 were pre-designed and ordered from IDT . A commercially available Gapdh probe was used as internal control . To ensure the specificity and efficiency of amplification, all assays were tested with serial dilutions of mouse cerebellum cDNA to verify efficiency (90%–110%) and linear amplification (R2 >0.96). mRNA levels were calculated by normalizing Ct values of relative genes to Gapdh.Sing-cell qPCR experiments were performed as previously described . Acute sAccelerating rotarod was carried out as previously reported . Mice weSocial interaction was evaluated in a three-chamber box. Mice were placed initially in the central chamber to allow 10 min habituation for all three chambers. For sociability session, a same sex- and age matched stranger mouse (stranger1) was placed inside an upside-down wire pencil cup in one of the side chambers. The other side had the same empty pencil cup. A test mouse was allowed 10 min to investigate the three chambers. For social novelty session, another stranger mouse (stranger2) was placed into the empty pencil cup and test mouse was allowed another 10 min to investigate between three chambers. The time mice spent in each chamber was recorded and analyzed using BIOBSERVE III tracking system.Mice were placed in the center of 40 × 40 cm white box and allowed to freely explore for 15 min. Videos were recorded and analyzed by BIOBSERVE III software. The 20 × 20 cm region in the center was defined as central zone. The total distance travelled and the activity exploring the center area were analyzed to evaluate the subject’s locomotor ability and anxiety levels.Beam-walk test was performed as previously described . BrieflyWe performed gait analysis as described previously . BrieflyExperiments and data analyses were performed blindly by coding viruses. Unpaired t-test or one-way ANOVA or two-way ANOVA or repeat measures ANOVA were used to analyze slice physiology data or immunohistochemistry data or behavior data as indicated in figure legends. Kolmogorov-Smirnov test was used to analyze the cumulative curves of mEPSCs or mIPSCs. Significance was indicated as *p < 0.05, **p < 0.01, ***p < 0.001. Data are expressed as means ± SEM. This study investigated the role of calsyntenin-3, an atypical cadherin, in controlling synaptic inputs to cerebellar Purkinje cells. It provides compelling evidence that elimination of calsyntenin-3 from cells in the cerebellar cortex alters the E/I balance for Purkinje cells, although Purkinje cell-specific manipulations were used in only some of the experiments. The results indicated that calsyntenin-3 increases the strength of excitatory parallel fiber inputs and decreases the strength of inhibitory inputs. public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by a Reviewing Editor and Gary Westbrook as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Calsyntenin-3, an atypical cadherin, suppresses inhibitory basket- and stellate-cell synapses but boosts excitatory parallel-fiber synapses in cerebellum" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this letter with what we consider essential revisions to help you prepare a revised submission. Most importantly, the authors need to show that Clstn3 is really the predominant Clstn3 expressed in Purkinje cells and that Clstn3 was specifically knocked out in Purkinje cells. These are the prerequisite conditions for the authors' conclusions as currently formulated.Essential revisions:A. Cell type specificity1) According to the in situ hybridization data , Clstn3 signals are detected in molecular and granular layers. I think that AAV-DJ using a U6 promoter to express gRNAs could infect various types of cells in the cerebellum. Since virus vectors were injected at various depths up to 2 mm, Clstn3 could be deleted in molecular-layer interneurons (MLI) that make inhibitory synapses onto Purkinje cells. Since Clstn3 could play roles in axonal transport, inhibitory synapses could have been impaired at least partly by presynaptic effects of Clstn3 in these cells. Clstn3 signals in granular layers, consisting of granule cells, Golgi cells and Lugaro cells, could also indirectly affect excitatory and inhibitory synapses in Purkinje cells. Thus, the authors need to ensure Purkinje-cell specific knockout of Clstn3 using specific promotors. Alternatively, the authors need to show that Clstn3 expression was unaffected in these neurons if the authors think that Clstn3 was specifically deleted in Purkinje cells by their AAV-DJ-U6 system.2) It would have been far better to selectively target Purkinje cells, but that was not done. For that reason, the authors need to do a thorough job of dealing with all of the potential complications. The Allen brain atlas shows that Clstn2 and Clstn3 are both expressed by Purkinje cells and that Clstn3 is also expressed by stellate cells and basket cells. The authors used a viral approach that did not select for Purkinje cells. As a result, the effects they see on synaptic transmission could arise from the elimination of all cells in a region rather than the selective elimination of calsyntenin-3 in Purkinje cells. This changes the interpretation of many of the experimental results. For this reason, it is crucial that the carefully determine whether Clstn2 is present in Purkinje cells, and whether Clstn3 is present in stellate cells and basket cells.3) An issue related to virus vectors is that although the authors used tdTomato as an expression marker of gRNAs (line 156), tdTomato driven by the CAG promoter does not necessarily ensure expression of gRNAs driven by the U6 promoter. Indeed, I feel that it is strange that AAV injected at various depths up to 2 mm did not reach the layer at all . I suspect that the 20% decrease in VGAT signals in the granular layer  may be caused by deletion of Clstn3 in granule cells, Golgi or Lugaro cells.4) According to the in situ hybridization data , Clstn2 is also expressed in Purkinje cells, weaking the authors' argument about the advantage of using these cells to prevent compensation by Clstns member. Thus, the authors need to show that the deletion of Clstn3 did not affect the amount and the localization of the Clstn2 in Purkinje cells. Immunoblot data shown in Figure S1A insufficient for this purpose.5) There is no description about how the authors examined off-target effects of CRISPR/Cas9 . It is completely unclear how they collected AAV-infected cells and determined the genome sequence. If they could show AAV-infected cells are Purkinje cells here, it would be strengthen the authors' argument about the cell-type specific knockout. Rescue of knockout phenotypes by re-introduction of gRNA-insensitive Clstn3 will be more helpful to rule out the off-target effects.B. Synapse numbers need to be assessed.1) Although the spine density is a reliable proxy for excitatory synapses in the hippocampus, it is not the case for Purkinje cells. Unlike the description in line 267, spines are known to be formed cell autonomously in the absence of presynaptic parallel fibers in Purkinje cells . For example, the number of dendritic spines is unaffected even in cbln1- and grid2-null mice, which showed severe reduction in the number of parallel fiber synapses. I think that the authors need to use pre- and postsynaptic markers.2) Counting the number of dendritic spines is not straightforward in Purkinje cells that extend numerous spines in all directions. I am not fully convinced by the morphological analyses of dendritic spines of Purkinje cells performed with Airyscan2. To address points 1) and 2), I would recommend using electron microscopic analyses, ideally with serial sections.3) For inhibitory synapses, what the authors showed was the changes in presynaptic markers. If the authors want to discuss synapse formation/maintenance, I think they should use pre- and postsynaptic markers to estimate the number of inhibitory "synapses."4) To strengthen the authors' conclusion about the specific role of Clstn3 in regulation of parallel-fiber synapses, they should show that Clstn3 is localized at parallel-fiber, but not climbing-fiber, synapses in wild-type Purkinje cells.C. Issues associated with behavioral experiments.1) Although the authors concluded that social interactions were not impaired in Clstn3 CRISPR KO mice (line 170), there is a tendency toward reduced sociability . The authors need to increase the number of animals to reach a conclusion.2) The behavioral data is not very compelling and is far from complete. It seems as if the rotarod assay was normal initially, and they just kept doing experiments until by trial there was a statistically significant difference. I think that this is of interest if it holds up, because it is a challenge to influence large parts of the cerebellum with AAV. I also think it is potentially very interesting, because the E/I balance seems to be strongly perturbed in these mice. Many other behavioral tests that are available to assess motor performance were not used. The number of experiments contributing to S3 is very small and it is not possible to conclude one way or anything regarding these behaviors. The number of experiments needs to be increased substantially or S3 should be eliminated.eLife. Your revised article has been evaluated by Gary Westbrook (Senior Editor). The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below. We appreciate that these issue do not affect the main conclusions, but these issues not to be addressed in the text before we can render a final decision.Thank you for resubmitting your work entitled "Calsyntenin-3, an atypical cadherin, suppresses inhibitory synapses but increases excitatory parallel-fiber synapses in cerebellum" for further consideration by Reviewer #2:I think the authors have thoroughly addressed comment A (Cell type specificity) of the Essential Revisions. Regarding comment B (Synapse numbers need to be assessed), there seems to be some misunderstanding. The point I wanted to make was that the spine density is NOT a reliable proxy for excitatory synapses in Purkinje cells. Indeed, in the review by Sudhof (2017) that the author cited, it is written as "Importantly, the loss of parallel-fiber synapses is NOT associated with a decrease in spine density." Thus, unlike in the hippocampus and cerebral cortex, the changes in the number of PF "synapses" are NOT reliably reflected by the number of Purkinje cell "spines." In other words, even if the number of dendritic spines is slightly increased, it is unclear whether they form synapses with PF terminals. The number of PF synapses can be counted easily by single-section EM analyses. However, considering that the major concern (comment A) has been thoroughly addressed by many additional experiments, I think authors could address comment B by rewording text. Similarly, the authors' responses to comment C  are OK.Reviewer #3:The authors have greatly improved the manuscript. They have done a good job of responding to most of the issues. In many cases their life would have been much easier had they simply taken a different experimental approach. For example, they could have done their manipulations selectively in Purkinje cells, but instead they had to do many controls to rule out alternative interpretations. In the end, they provide compelling data that supports their main conclusions. In so doing they have established that in cerebellar Purkinje cells, the acute knockout of calsyntenin-3 increases granule cell synapse density and decreases inhibitory synapse density. This is the first study showing that a particular synaptic adhesion molecule differentially regulates excitatory (increased) and inhibitory (decreased) synapses.1. As stated in the initial review, the authors have a tendency to draw conclusions that go beyond their data. They responded these issues by stating that it was a standard in the field, based on a previous study that took this approach.- Specifically, when asked about the 0-60 and >60 analysis they stated that it was standard in the field. While there may be some papers that adopt this approach, I do not think it is a justified approach. I do not agree that it can be used to discriminate between stellate cell and basket cell inputs. They should not use such a poorly justified approach simply because it has been used by others.-The same approach was taken in response to the 1ms rise time being used to distinguish between parallel fiber and climbing fiber mEPSCs. They state "The claim that the rise time of 1 ms distinguishes between climbing- and parallel-fiber mEPSCs is not ours, but well documented in the literature . We agree that it is likely only an approximation, but as an approximation we believe -as do others- that it is useful." They may believe it, but I think it is very weak and if they do use it they need point out how flimsy the evidence is for this. Finding a paper that uses this approach and turning into a standard for the field is not something I agree with.2. They were asked for an explanation of how they determined the capacitance and membrane resistances, rather than simply providing the details of the methods, they gave a long and not very helpful explanation. Please describe the methods in detail. The issue of active conductances, including hyperpolarization-activated cation current, could certainly can come into play. The passive properties of the PCs are very important for the interpretation of the data, so detailed methods are required.3. They were asked to strengthen the authors' conclusion about the specific role of Clstn3 in regulation of parallel-fiber synapses, they should show that Clstn3 is localized at parallel-fiber, but not climbing-fiber, synapses in wild-type Purkinje cells. They responded: As cited in the manuscript, Clstn3 has already been localized to parallel-fiber synapses in the cerebellum by immuno-EM in published papers. This does not address the issue of whether Clstn3 is absent from climbing fiber synapses.eLife. Your revised article has been evaluated by Gary Westbrook (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled "Calsyntenin-3, an atypical cadherin, suppresses inhibitory synapses but increases excitatory parallel-fiber synapses in cerebellum" for further consideration by The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:Essential revisions:The editor and reviewers acknowledge that the authors made a real effort to address the previous reviews and that a large amount of work is included in the manuscript. However there are some remaining issues that we think need to be addressed as follows based on the re-reviews and the discussion between editors and reviewers:1. The sparse lentivirus expression experiments are a great addition that satisfies many concerns for a subset of experiments. However, these additions raise serious concerns about the interpretation of all the experiments that were not studied using sparse labeling. The new results establish that the original experiments could be directly affecting MLIs and granule cells when many cells in a region are targeted. It must be assumed that direct effects on granule cells and MLIs contribute to any effects they describe, whether it be behavior or electrophysiology. The control experiments using sparse expression are only useful for those specific experiments that were performed. It is only valid to present data in which they have shown sparse and dense viral targeting show quantitatively the same result. Other data are uninterpretable at this point. The authors seem to have used the new experiments as a blanket control experiment to justify the interpretation of all experiments as being attributable to Clstn2 in Purkinje cells. This is drawing conclusions that go beyond what is supported by the data. The authors must be more careful in drawing conclusions from what is supported by the data.2. Overall the behavioral experiments are weak. One basic issue is that a lack of effect could be attributed to not affecting all of the Purkinje cells that contribute to a behavior. The second is that the approach used for behavioral studies leaves open the possibility that off target effects could be responsible for the behavioral effects (as in point 1). There are good Cre lines for Purkinje cells. It would have been straightforward to selectively target Purkinje cells and that is what should have been done. The authors decided against using this approach and did not address the use of this approach in their response. Without selectively targeting Purkinje cells, behavioral disruptions are basically uninterpretable and the authors must acknowledge these limitations.3. The authors imply that Clstn3 expression at higher levels in Purkinje cells than in other cells suggests that it means that Clstn3 is unlikely to be important in other cell types. This is not convincing logic as there certainly seems to be appreciable Clstn3 in other cell types. The high level of Clstn3 in granule cells and basket cells highlights the problem in interpreting many of their experiments. The authors need to adjust their conclusions in this regard, short of redesigning experiments that specifically target Purkinje cells.eLife paper  in which the elimination of granule cells and their associated presynaptic boutons, albeit in mature mice, left behind Purkinje cells covered with normal looking spines that lacked presynaptic boutons. Even if the spines are formed initially, they remain in the adults. Overall, the spine density is NOT a reliable proxy for excitatory synapses in Purkinje cells. Indeed, in the review by Sudhof (2017) that the author cited, it is written as "Importantly, the loss of parallel-fiber synapses is NOT associated with a decrease in spine density." Thus, unlike in the hippocampus and cerebral cortex, the changes in the number of PF "synapses" are NOT reliably reflected by the number of Purkinje cell "spines." In other words, even if the number of dendritic spines is slightly increased, it is unclear whether they form synapses with PF terminals. Thus, spine density is a flawed measure for excitatory synapses that should be acknowledged.4. In the response on page 6 the authors dismiss the prevalence of naked spines, which disregards a recent  The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this letter with what we consider essential revisions to help you prepare a revised submission. Most importantly, the authors need to show that Clstn3 is really the predominant Clstn3 expressed in Purkinje cells and that Clstn3 was specifically knocked out in Purkinje cells. These are the prerequisite conditions for the authors' conclusions as currently formulated.In the revised paper, we have performed multiple experiments and data analyses to address this point. Specifically:1) We have analyzed single-cell RNAseq data of the cerebellum which demonstrate that in the cerebellum, Purkinje cells uniquely express Clstn3, and that Clstn3 is not expressed at high levels in other cerebellar cells .2) We have performed quantitative RT-PCR analyses of cytosol aspirated from Purkinje cells and other neurons with primers that were validated specifically for this purpose to demonstrate that Purkinje cells express Clstn3 at > 50-fold higher levels than Clstn1 or Clstn2, and that the levels of Clstn3 are much lower in other cerebellar cells .3) We analyzed Purkinje cells after the Clstn3 deletion using single-cell qRT-PCR with pipette-aspirate cytosol, and demonstrate that only Clstn3 but not other calsyntenins are suppressed by CRISPR-mediated deletion .Together, we believe that these results conclusively demonstrate that a) Purkinje cells express Clstn3 at much higher levels than other cerebellar cells, and b) other cerebellar cells do not express Clstn3 at similarly high levels.Essential revisions:A. Cell type specificity1) According to the in situ hybridization data , Clstn3 signals are detected in molecular and granular layers. I think that AAV-DJ using a U6 promoter to express gRNAs could infect various types of cells in the cerebellum. Since virus vectors were injected at various depths up to 2 mm, Clstn3 could be deleted in molecular-layer interneurons (MLI) that make inhibitory synapses onto Purkinje cells. Since Clstn3 could play roles in axonal transport, inhibitory synapses could have been impaired at least partly by presynaptic effects of Clstn3 in these cells. Clstn3 signals in granular layers, consisting of granule cells, Golgi cells and Lugaro cells, could also indirectly affect excitatory and inhibitory synapses in Purkinje cells. Thus, the authors need to ensure Purkinje-cell specific knockout of Clstn3 using specific promotors. Alternatively, the authors need to show that Clstn3 expression was unaffected in these neurons if the authors think that Clstn3 was specifically deleted in Purkinje cells by their AAV-DJ-U6 system.We agree that in our experiments, the AAVs we use likely infect other neurons in addition to Purkinje cells, and that in these neurons Clstn3 expression will also be suppressed. Thus, we concur that it is conceivable that the phenotype we observed could also be due to a suppression of Clstn3 in other cerebellar neurons, and that our data did not conclusively rule out the possibility of a presynaptic function of Clstn3 in excitatory granule cells or in inhibitory basket/stellate cells in the cerebellum. To address this concern, we have performed two sets of new experiments:1) We have used sparse lentiviral infections to delete Clstn3 only in a few isolated Purkinje cells of the entire cerebellum of an animal. In these experiments, a single Purkinje cell is infected in a broad section of the cerebellar cortex . We then demonstrate that with these sparse Clstn3 deletions, the Purkinje cells exhibit the same phenotype as with the broad deletions of the entire cerebellar Clstn3 using AAVs. Thus, Clstn3 acts cell-autonomously in Purkinje cells . Note that in these sparse deletions of Clstn3, we observe the identical and intriguing increase in parallel-fiber EPSCs and suppression of basket/stellate cell IPSCs as in AAV-mediated Clstn3 deletions. This experiment is better than the suggested experiment with a Purkinje cell-specific promoter because in viral vectors, cell type-specific promoters exhibit limited specificity, and only transgenic methods are suitable to achieve true cell type specificity.2) In parallel, we have analyzed the effect of the sparse deletion of Clstn3 in Purkinje cells on the expression of Clstn1, Clstn2, and Clstn3 in Purkinje cells and in surrounding neurons . Because the lentiviral infections were so sparse, these were very difficult experiments with only a few cells per animal. Nevertheless, the data show that the sparse deletion of Clstn3 in Purkinje cells only lowers Clstn3, but not Clstn1 or Clstn2, levels in Purkinje cells, and that it only lowers Clstn3 levels in Purkinje cells but not in surrounding basket or granule cells . We believe that this experiment conclusively demonstrates that it is truly the postsynaptic deletion of Clstn3 that causes the robust phenotype we observe, which is even more impactful considering that the KO is unlikely to be 100% effective since it is not a conditional deletion.3) Finally, we would like to note that given the relatively specific expression of Clstn3 in Purkinje cells compared to other cerebellar neurons, it is unlikely that the Clstn3 deletion operates in these other neurons, although we do agree that without the new experiments we just described above, we could not have ruled out this unlikely possibility.We hope that these new data will be persuasive in showing that it is truly the deletion of postsynaptic Clstn3 that produces the strong phenotype we observe.2) It would have been far better to selectively target Purkinje cells, but that was not done. For that reason, the authors need to do a thorough job of dealing with all of the potential complications. The Allen brain atlas shows that Clstn2 and Clstn3 are both expressed by Purkinje cells and that Clstn3 is also expressed by stellate cells and basket cells. The authors used a viral approach that did not select for Purkinje cells. As a result, the effects they see on synaptic transmission could arise from the elimination of all cells in a region rather than the selective elimination of calsyntenin-3 in Purkinje cells. This changes the interpretation of many of the experimental results. For this reason, it is crucial that the carefully determine whether Clstn2 is present in Purkinje cells, and whether Clstn3 is present in stellate cells and basket cells.This are essentially the same concerns as expressed in the previous two comments, namely the fact that Clstn3 is also expressed in other neurons besides Purkinje cells, that Purkinje cells could also express other calsyntenins, and that the AAV infection of the cerebellar cortex could also have deleted Clstn3 in other neurons. These concerns were addressed in the new experiments and analyses described above.3) An issue related to virus vectors is that although the authors used tdTomato as an expression marker of gRNAs (line 156), tdTomato driven by the CAG promoter does not necessarily ensure expression of gRNAs driven by the U6 promoter. Indeed, I feel that it is strange that AAV injected at various depths up to 2 mm did not reach the layer at all . I suspect that the 20% decrease in VGAT signals in the granular layer  may be caused by deletion of Clstn3 in granule cells, Golgi or Lugaro cells.This comment voices three concerns that we have addressed as follows:a) The concern that tdTomato expression by a virus via the CAG promoter does not guarantee expression of the gRNA via the U6 promoter from the same virus. We completely agree, which is why we carefully demonstrate that Clstn3 mRNAs are severely suppressed by the gRNA as shown by multiple types of measurements .b) The concern that it is strange that AAVs injected into the cerebellar cortex apparently did not reach the upper layers of the cerebellar cortex. The original Figure 2E  did indeed appear to suggest that the upper layers of the cerebellar cortex are not infected, but we have now added a new supplementary figure  to show that they are. We think that part of the confusion here derives from the way we designed our experiments, which use tdTomato expression from the virus as an indicator, NOT Cre-expression by the virus in a Cre-dependent tdTomato indicator mouse. In the latter case, very low levels of Cre expression are sufficient to elicit a tdTomato signal which is uniform across the infected tissue. This can be misleading. In our case of virally expressed tdTomato, the levels of tdTomato reflect the viral titer, and it is to be expected that these levels are highest closer to the injection sites, and lower at more distant sites.c) The concern that the loss of inhibitory synapses via the cerebellar CRISPR-mediated deletion of Clstn3 is due to a suppression of Clstn3 expression in inhibitory neurons where Clstn3 might have a presynaptic function instead of the suppression of Clstn3 expression in Purkinje cells. This valid concern is the same concern voiced in previous comments, and was addressed above with the sparse deletion of Clstn3 only in Purkinje cells and the demonstration that Clstn3 is expressed at high levels only in Purkinje cells.4) According to the in situ hybridization data , Clstn2 is also expressed in Purkinje cells, weaking the authors' argument about the advantage of using these cells to prevent compensation by Clstns member. Thus, the authors need to show that the deletion of Clstn3 did not affect the amount and the localization of the Clstn2 in Purkinje cells. Immunoblot data shown in Figure S1A insufficient for this purpose.Agreed – we have addressed this concern with the following new data:a) Single-cell qRT-PCR analyses of wild-type mice demonstrate that Clstn3 is expressed at >25-fold higher levels in Purkinje cells than Clstn1 or Clstn2 .b) Single-cell qRT-PCR analyses of sparsely infected Purkinje cells show that the CRISPR deletion affects only Clstn3 levels but not Clstn1 or Clstn2 .c) Analyses of single-cell RNAseq data deposited by the McCarroll lab show that, consistent with our qRT-PCR studies, Purkinje cells express only low amounts of Clstn1 or Clstn2, which are, however, expressed at higher levels in other cerebellar cells .5) There is no description about how the authors examined off-target effects of CRISPR/Cas9 . It is completely unclear how they collected AAV-infected cells and determined the genome sequence. If they could show AAV-infected cells are Purkinje cells here, it would be strengthen the authors' argument about the cell-type specific knockout. Rescue of knockout phenotypes by re-introduction of gRNA-insensitive Clstn3 will be more helpful to rule out the off-target effects.We apologize for the lack of clarity and have improved this in the revised paper. Please note that we analyzed the off-target effects of our CRISPR manipulations using bioinformatics and genomic PCRs, but did not perform whole-genome sequencing to confirm these analyses (which would be another large project). We concur that rescue experiments would be useful, but they exceed the scope of the current study, which is already a massive project spanning many years of work and now amounting to 11 data figures. Please also note that we have now addressed the cell-type specificity with the sparse lentiviral infection as described above.B. Synapse numbers need to be assessed.1) Although the spine density is a reliable proxy for excitatory synapses in the hippocampus, it is not the case for Purkinje cells. Unlike the description in line 267, spines are known to be formed cell autonomously in the absence of presynaptic parallel fibers in Purkinje cells . For example, the number of dendritic spines is unaffected even in cbln1- and grid2-null mice, which showed severe reduction in the number of parallel fiber synapses. I think that the authors need to use pre- and postsynaptic markers.The reviewer expresses a view of the ‘naked spines’ in GluD2 and Cbln1 mutant mice that is still written in many textbooks, but that is simply incorrect. Claudio Sotelo was a giant in the field of cerebellar anatomy, but much has been done since. Spines are NOT known to be formed cell-autonomously – the famous ‘naked spines’ in the Cbln1 and Grid2 mutant mice arise because synapses are initially formed normally, but then degenerate (please see the discussion in Sudhof (2017) for details). What happens, simply, is that defective synaptic transmission leads to a degradation of nerve terminals first, and then spines. Spine numbers are not normal but decreased in these mutants, and the prevalence of ‘naked spines’ is rare. An extensive and beautiful literature, primarily from leading Japanese labs in studies published from 1990 to 2010, documents this .2) Counting the number of dendritic spines is not straightforward in Purkinje cells that extend numerous spines in all directions. I am not fully convinced by the morphological analyses of dendritic spines of Purkinje cells performed with Airyscan2. To address points 1) and 2), I would recommend using electron microscopic analyses, ideally with serial sections.We agree that 3D reconstructions using serial EM sections would be great and appreciate the recommendation to perform such analyses for wild type and mutant cerebellum. However, this has not yet been achieved for even the wild-type cerebellum – it would basically represent a >5-year project given the large size of the Purkinje cell dendritic arbors and the enormous degree of connectivity of parallel fiber synapses. BUT: We did perform full 3D reconstructions by confocal microscopy, using advanced microscopes and highly respected software! Although these reconstructions took months to accomplish for a limited number of cells, the results are unequivocal and represent the state-of-the-art in this field. Thus, we did not just count spines in sections, but performed actual reconstructions that enable counting numerous spines in all directions.3) For inhibitory synapses, what the authors showed was the changes in presynaptic markers. If the authors want to discuss synapse formation/maintenance, I think they should use pre- and postsynaptic markers to estimate the number of inhibitory "synapses."In new experiments we have measured the density of inhibitory synapses using antibodies to GABA-Aa1 receptors in matched control and Clstn3-deficient cerebellar cortex . The results confirm the previous conclusions, which are also supported by the measurements of mIPSCs.4) To strengthen the authors' conclusion about the specific role of Clstn3 in regulation of parallel-fiber synapses, they should show that Clstn3 is localized at parallel-fiber, but not climbing-fiber, synapses in wild-type Purkinje cells.As cited in the manuscript, Clstn3 has already been localized to parallel-fiber synapses in the cerebellum by immuno-EM in published papers.C. Issues associated with behavioral experiments.1) Although the authors concluded that social interactions were not impaired in Clstn3 CRISPR KO mice (line 170), there is a tendency toward reduced sociability . The authors need to increase the number of animals to reach a conclusion.As recommended, we have greatly expanded the behavioral analyses of the mice in which cerebellar Clstn3 was deleted, including more exhaustive analysis of social interactions. The new data are now shown in Figure 3.2) The behavioral data is not very compelling and is far from complete. It seems as if the rotarod assay was normal initially, and they just kept doing experiments until by trial there was a statistically significant difference. I think that this is of interest if it holds up, because it is a challenge to influence large parts of the cerebellum with AAV. I also think it is potentially very interesting, because the E/I balance seems to be strongly perturbed in these mice. Many other behavioral tests that are available to assess motor performance were not used. The number of experiments contributing to S3 is very small and it is not possible to conclude one way or anything regarding these behaviors. The number of experiments needs to be increased substantially or S3 should be eliminated.The protocol we use for the rotarod assay is a standard method using an escalating challenge. This protocol is not an afterthought, but was described in detail in our Rothwell et al., 2014 paper as cited. The protocol was developed to measure motor learning, which is clearly impaired in our mice. We have now expanded the behavioral analysis and show this analysis in the new Figure 3. The bottom line is that a discrete impairment of motor behaviors can be reproducibly observed with no changes in other behavioral parameters.Reviewer #2:I think the authors have thoroughly addressed comment A (Cell type specificity) of the Essential Revisions. Regarding comment B (Synapse numbers need to be assessed), there seems to be some misunderstanding. The point I wanted to make was that the spine density is NOT a reliable proxy for excitatory synapses in Purkinje cells. Indeed, in the review by Sudhof (2017) that the author cited, it is written as "Importantly, the loss of parallel-fiber synapses is NOT associated with a decrease in spine density." Thus, unlike in the hippocampus and cerebral cortex, the changes in the number of PF "synapses" are NOT reliably reflected by the number of Purkinje cell "spines." In other words, even if the number of dendritic spines is slightly increased, it is unclear whether they form synapses with PF terminals. The number of PF synapses can be counted easily by single-section EM analyses. However, considering that the major concern (comment A) has been thoroughly addressed by many additional experiments, I think authors could address comment B by rewording text. Similarly, the authors' responses to comment C  are OK.We agree that spine numbers are not a completely reliable proxy for synapse numbers, although in most cases quite accurate. However, but not always a reliable one since there are situations in which pre- and postsynaptic specializations diverge. However, since we also measured synapse numbers by immunocytochemistry (which has its own acknowledged limitations), we believe our conclusions are justified. We have now made this point clearer in the paper.Reviewer #3:The authors have greatly improved the manuscript. They have done a good job of responding to most of the issues. In many cases their life would have been much easier had they simply taken a different experimental approach. For example, they could have done their manipulations selectively in Purkinje cells, but instead they had to do many controls to rule out alternative interpretations. In the end, they provide compelling data that supports their main conclusions. In so doing they have established that in cerebellar Purkinje cells, the acute knockout of calsyntenin-3 increases granule cell synapse density and decreases inhibitory synapse density. This is the first study showing that a particular synaptic adhesion molecule differentially regulates excitatory (increased) and inhibitory (decreased) synapses.We appreciate the positive comments.1. As stated in the initial review, the authors have a tendency to draw conclusions that go beyond their data. They responded these issues by stating that it was a standard in the field, based on a previous study that took this approach.- Specifically, when asked about the 0-60 and >60 analysis they stated that it was standard in the field. While there may be some papers that adopt this approach, I do not think it is a justified approach. I do not agree that it can be used to discriminate between stellate cell and basket cell inputs. They should not use such a poorly justified approach simply because it has been used by others.We realize that the reviewer is deeply concerned about the approach of classifying mIPSCs via their amplitude, and that he/she feels that this approach is wrong even though it has been used by others. To accommodate this concern, we have now reworded the description of this analysis in the paper to exclude the claim that the mIPSCs with different amplitudes are differentially derived from basket and stellate cells, and only use this classification to show that the phenotype we observe persists in different subsets of mIPSCs identified by their differential amplitudes. We mention that some people in the field believe that these different amplitudes correspond to different neuronal origins, but make no claim that this is necessarily a fact. We hope the reviewer will agree with these very cautious descriptions.-The same approach was taken in response to the 1ms rise time being used to distinguish between parallel fiber and climbing fiber mEPSCs. They state "The claim that the rise time of 1 ms distinguishes between climbing- and parallel-fiber mEPSCs is not ours, but well documented in the literature . We agree that it is likely only an approximation, but as an approximation we believe -as do others- that it is useful." They may believe it, but I think it is very weak and if they do use it they need point out how flimsy the evidence is for this. Finding a paper that uses this approach and turning into a standard for the field is not something I agree with.We also agree that using the criterion of the mEPSC kinetics is an imperfect approach to distinguishing between parallel- and climbing-fiber synapses. However, based on classical studies in the field, such as the Magee and Cook 2008 paper that beautifully documents the relation of mEPSC kinetics to dendritic distance, we do feel this criterion has some validity. To assuage the reviewer’s concerns and follow her/his recommendation, we have now emphasized even more strongly in the paper that this classification of mEPSCs is only an approximation, not a definitive measurement.2. They were asked for an explanation of how they determined the capacitance and membrane resistances, rather than simply providing the details of the methods, they gave a long and not very helpful explanation. Please describe the methods in detail. The issue of active conductances, including hyperpolarization-activated cation current, could certainly can come into play. The passive properties of the PCs are very important for the interpretation of the data, so detailed methods are required.We have now added a detailed methods description in the revised manuscript.3. They were asked to strengthen the authors' conclusion about the specific role of Clstn3 in regulation of parallel-fiber synapses, they should show that Clstn3 is localized at parallel-fiber, but not climbing-fiber, synapses in wild-type Purkinje cells. They responded: As cited in the manuscript, Clstn3 has already been localized to parallel-fiber synapses in the cerebellum by immuno-EM in published papers. This does not address the issue of whether Clstn3 is absent from climbing fiber synapses.As cited in the manuscript, previous studies localized Clstn3 to parallel-fiber but not climbing-fiber synapses. However, this finding does not rule out a low concentration of Clstn3 in climbing-fiber synapses. We cannot exclude the presence of some Clstn3 molecules from climbing-fiber synapses, nor can anyone else for that matter, because any immunocytochemical or imaging technique will never be sensitive enough to rule out levels of a synaptic molecule in a synapse if it present in the same neurons at other synapses at much higher levels. We strongly believe that the lack of an effect of the Clstn3 KO on climbing-fiber synaptic transmission shows that Clstn3 has no major function at these synapses. We believe this is conclusive, and that most people in the field would agree, no matter whether or not low concentrations of Clstn3 might be present in climbing-fiber synapses. We have now added a sentence to that effect in the paper.Essential revisions:The editor and reviewers acknowledge that the authors made a real effort to address the previous reviews and that a large amount of work is included in the manuscript. However there are some remaining issues that we think need to be addressed as follows based on the re-reviews and the discussion between editors and reviewers:1. The sparse lentivirus expression experiments are a great addition that satisfies many concerns for a subset of experiments. However, these additions raise serious concerns about the interpretation of all the experiments that were not studied using sparse labeling. The new results establish that the original experiments could be directly affecting MLIs and granule cells when many cells in a region are targeted. It must be assumed that direct effects on granule cells and MLIs contribute to any effects they describe, whether it be behavior or electrophysiology. The control experiments using sparse expression are only useful for those specific experiments that were performed. It is only valid to present data in which they have shown sparse and dense viral targeting show quantitatively the same result. Other data are uninterpretable at this point. The authors seem to have used the new experiments as a blanket control experiment to justify the interpretation of all experiments as being attributable to Clstn2 in Purkinje cells. This is drawing conclusions that go beyond what is supported by the data. The authors must be more careful in drawing conclusions from what is supported by the data.Cbln1 KO, de Zeuw’s pioneering work on GluA1, and Mishina’s discoveries of GluD2. All of this with one sweep would be uninterpretable because none of these studies used cell-type specific manipulations. These authors argued previously, we think convincingly, that the context of their experiments allow definitive interpretable conclusions, and we hope that the same applies to our experiments.We regret to say that we disagree with this comment, which may have been due to our inability to clearly communicate the logic of our experiments. The reviewer(s) offers the fundamental argument that most of our experiments are uninterpretable because they were performed by manipulations that are not cell type-specific. If this argument was correct, the vast majority of the literature on synaptic mechanisms on the cerebellum would have to be dismissed, including Yuzaki’s classical studies on the To recap, our fundamental observations are that (a) the acute Calsyntenin-3 deletion using in vivo CRISPR introduced by AAVs in the cerebellar cortex caused an increase in parallel-fiber synapse numbers and a decrease in GABAergic synapses on Purkinje cells, as documented by multiple electrophysiological measurements, staining for a panoply of synaptic markers, and reconstruction of Purkinje cells; (b) Calsyntenin-3 is expressed at much higher levels in Purkinje cells than in other neurons or glia of the cerebellum, such that granule cells express >10-fold lower levels and basket cells >5-fold lower levels of Calsyntenin-3 than Purkinje cells; (c) the AAV stain that we use infects Purkinje cells preferentially; and (d) the sparse Calsyntenin-3 deletion in Purkinje cells using lentiviruses causes the same electrophysiological phenotype as the deletion of Calsyntenin-3 in many Purkinje cells by AAVs.Cbln1 deletion, the effect sizes we see are large, and surely Calsyntenin-3 emerges here as a significant contributor to shaping cerebellar circuits.Based on these combined findings, we conclude that Calsyntenin-3 functions both to promote and to restrict synapse formation, and that Calsyntenin-3 acts in Purkinje cells. We did not rule out a contribution of Calsyntenin-3 to the function of other cerebellar neurons. We would like to argue that the findings speak for themselves: postsynaptic Calsyntenin-3 in Purkinje cells acts as a major regulator of inhibitory basket/stellate-cell and or parallel-fiber synapses. Compared for example to the We have modified the text to make sure that the argument we make is clearly articulated, and hope this will now be acceptable to the scientific community and to the reviewers.2. Overall the behavioral experiments are weak. One basic issue is that a lack of effect could be attributed to not affecting all of the Purkinje cells that contribute to a behavior. The second is that the approach used for behavioral studies leaves open the possibility that off target effects could be responsible for the behavioral effects (as in point 1). There are good Cre lines for Purkinje cells. It would have been straightforward to selectively target Purkinje cells and that is what should have been done. The authors decided against using this approach and did not address the use of this approach in their response. Without selectively targeting Purkinje cells, behavioral disruptions are basically uninterpretable and the authors must acknowledge these limitations.Overall the behavioral experiments are weak … Without selectively targeting Purkinje cells, behavioral disruptions are basically uninterpretable and the authors must acknowledge these limitations”. We agree that the behavioral experiments are not extensive, but our paper is not intended as a major behavioral study, and the behavioral data occupy only one of 11 main and 8 supplementary figures. Nevertheless, the behavioral phenotype we observe is robust and consistent with a cerebellar dysfunction. It is conceivable that not all Purkinje cells were infected by our in vivo manipulations, but the presence of a major phenotype appears to us to be sufficient to argue that the physiological changes we observe are behaviorally relevant, which is the purpose of the behavioral experiments. The fact that the KO of a gene that is expressed primarily in Purkinje cells using a viral CRISPR manipulation that primarily affects Purkinje cells causes a major phenotype means to us that this gene is important for cerebellar functions, and that its effects are likely mediated by Purkinje cells.This comment is similar to the first comment, and again may be partly due to our inability to communicate facts clearly, for which we apologize. The comment concludes that “We have modified the text to make sure that all readers understand that our study uses behavioral experiments as a way to document that the Calsyntenin-3 deletion in the cerebellum produces a biologically significant phenotype, and that we do not present an exhaustive behavioral analysis, although that might be apparent from the data.3. The authors imply that Clstn3 expression at higher levels in Purkinje cells than in other cells suggests that it means that Clstn3 is unlikely to be important in other cell types. This is not convincing logic as there certainly seems to be appreciable Clstn3 in other cell types. The high level of Clstn3 in granule cells and basket cells highlights the problem in interpreting many of their experiments. The authors need to adjust their conclusions in this regard, short of redesigning experiments that specifically target Purkinje cells.Again, we fear that we were not clear in our presentation since the reviewer states that there are high levels of Calsyntenin-3 in granule and basket cells, which is not what we show or what others have found. Our results and published in situ and RNAseq data show that Calsyntenin-3 expression in granule and basket cells is very low – in our in situ experiments, we cannot even detect Calsyntenin-3 in granule cells, only sensitive RT-PCR assays enable that.Moreover, we have now made sure in the paper that we make no claims that Calsyntenin-3 is unlikely to be important in other cell types. Since we did not study other cerebellar cell types, we don’t know. Our conclusion is that Calsyntenin-3 in Purkinje cells regulates the formation/maintenance of parallel-fiber and basket/stellate cell synapses on the Purkinje cells. We do believe this conclusion is justified since we show (i) that Calsyntenin-3 is primarily expressed in Purkinje and cells, (ii) that a global viral manipulation that primarily if not exclusively affects Purkinje cells causes a large phenotype as assessed by a broad number of assays, and (iii) that a sparse manipulation of the same gene only in Purkinje cells replicates the electrophysiological phenotype of the global Purkinje cell deletion. We have now re-formulated our text to reflect this more clearly.4. In the response on page 6 the authors dismiss the prevalence of naked spines, which disregards a recent eLife paper  in which the elimination of granule cells and their associated presynaptic boutons, albeit in mature mice, left behind Purkinje cells covered with normal looking spines that lacked presynaptic boutons. Even if the spines are formed initially, they remain in the adults. Overall, the spine density is NOT a reliable proxy for excitatory synapses in Purkinje cells. Indeed, in the review by Sudhof (2017) that the author cited, it is written as "Importantly, the loss of parallel-fiber synapses is NOT associated with a decrease in spine density." Thus, unlike in the hippocampus and cerebral cortex, the changes in the number of PF "synapses" are NOT reliably reflected by the number of Purkinje cell "spines." In other words, even if the number of dendritic spines is slightly increased, it is unclear whether they form synapses with PF terminals. Thus, spine density is a flawed measure for excitatory synapses that should be acknowledged.We also apologize if we created the impression that we dismissed the possibility of naked spines, which was not our intention, and have now mentioned this possibility extensively and multiple times throughout the paper. But we have to confess that we do not quite understand the reviewers’ criticism here since the previous version already mentioned naked spines and explicitly said that they might contribute to the spine density, and that measurements of the spine density are only interpretable in the context of multiple approaches that probe synapse density.slight’ but is the biggest ever observed as far as we could tell) by itself does not prove that there are more parallel-fiber synapses, but we do not claim this – we just use it as one of many measurements. It should also be noted that the naked spines do represent remnants of previous synapses – so they do reflect synapses that once existed – and that no condition every induced an increase in naked spines. We have modified the text to make sure that no reader feels we dismiss naked spines, and that all readers understand the multiple parameters we measured to assess parallel-fiber synapse numbers.In our experiments, we use the number of spines as one of 6 measurements to assess the density of excitatory parallel-fiber synapses. These measurements include in addition to spine density measurements: quantitative immunocytochemistry for vGluT1, GluA1, GluA2, mEPSCs, and evoked parallel-fiber EPSCs. It seems to us that, with a complete concordance of all six measurements, we are on firm grounds to conclude that parallel-fiber synapse numbers are increased. We agree that the increase in spines (which the reviewer calls ‘


Key features include guidance to set goals, self-monitor and assess achievements, and experiential learning via the connected activity trackers which allows for continuous steps recording during the program. iEngage was implemented in two schools over 5 weeks with 10–12 years old adolescents (n = 57) and PA outcomes compared to control group (n = 26). Results show that adolescents successfully set goals and self-assessed achievements during the program, progressing toward higher physical activity (PA) levels as shown by the 30% increase in daily steps through the program  with boys increasing goals and achievements faster than girls. The consistency in days totalling at least 11,000 steps/day increased from 35% at the start to 48% at the end of the program. The increase in PA is confirmed through the assessment of MVPA during schooldays pre- and post- program via research grade wrist accelerometers in both iEngage and control participants. Contrasting with the control group, MVPA was increased in the week following the program  in short bouts, particularly during lunch time, recess and after school. This study shows that a digital program integrating activity trackers data, health education, goals setting and self-monitoring of PA, helped young adolescents enhance PA goals, improve achievements and increase MVPA.iEngage is a modular health education and behavioural change program designed to help adolescents increase moderate to vigorous physical activity (MVPA). The program is delivered through the iEngage app which integrates activity trackers data (Misfit Ray Physical activity (PA) is a major component of health and wellbeing across the lifespan and contributes to reducing all-cause mortality. However, current evidence from large population cross-sectional surveys indicates that 81% of children and adolescents globally do not participate in sufficient amounts of PA . SurveysBecause low participation in PA is a risk factor for the development of overweight and obesity in young people, leading to adverse cardiometabolic profiles, there is an urgent need to identify approaches and interventions that are effective , 6. For Numerous interventions have attempted to enhance participation in PA and MVPA in children and adolescents; most of them showing no or small changes in PA behaviour in response to interventions, indicating that changing PA behaviour in adolescents is challenging . School-In this article, we present our approach to designing and implementing iEngage, an educational and behavioural change program tailored to 10–12 years old adolescents. Building on an initial work , the proWe hypothesised that iEngage, combining experiential learning and behavioural change techniques, delivered via a user-friendly digital environment with integrated activity trackers, will be able to help adolescents enhance PA behaviour.We present the original design approach of the iEngage program and report on its effectiveness for enhancing PA goals and achievements and increase MVPA in daily life of 10–12-year-old Australian adolescents.iEngage is an evidence-based health education and behavioural change program designed to help young adolescents increase MVPA. The program is supported by a digital environment that includes the iEngage app connected to activity trackers. Using live data from these trackers and digital health education modules, iEngage aims to drive behavioural change through experiential learning, goal setting and self-assessment of achievements. The program and companion app were co-designed with experts in physical education, exercise science, data science and mobile health industry experts. A few short sessions were organised to gather feedback from adolescents.Topics such as health, intensity of PA, MVPA, sedentary behaviours (SB), physical fitness, physical effort, exertion, mode of exercise or the impact of PA on body functions are explained through a series of 10 digital modules  while aciEngage also aligns with the Australian Physical Literacy Framework. Physical literacy is described as the skills, knowledge and behaviours that help us lead active lives , 17, 18.amount of daily steps and MVPA they aim for  and (2) choosing a mission from four suggestions . To facilitate the quantitative goal, the app proposes a finite list of increasing commitments to choose from, with increasing boundaries throughout the program. Step choices from modules 1 to 10 are: 5,000 to 7,000 steps in module 1; 6,000 to 10,000 steps in module 2 and so on. MVPA choices start from module 6 and increase from 20 min to 60-min daily of MVPA as follows: 9000 steps/day and 20 min of MVPA, 10000 steps/day and 30 min of MVPA, 11,000 steps/day and 40 min of MVPA, 12000 steps/day and 60 min of MVPA. The maximum number of steps is based on studies estimating that performing 12,000 steps/day is possible only if adolescents engage in at least 45 minutes of MVPA [The process for personalised goal setting and assessment of achievements is designed as follows: in each module, the adolescent must set two types of goals for the next 3 days through two tasks: (1) choosing the The missions encourage adolescents to engage with the program through diverse activities with their parents or siblings, as research shows the importance of family, friends and community in driving behaviour change in adolescents . Example(C)) in the iEngage app; 2) reporting achievements in the app, 3) providing a self-assessment of achievements by answering a quiz and 4) reporting on completion of their mission. It is made clear in module 1 that iEngage focuses on progressing and achieving individual goals. iEngage has no component involving ranking the adolescents’ achievements and it has no competitive aspect. This is aligned with studies showing that competition and performance-based ranking can lead to adverse outcomes in adolescents [In each module but the first one, the adolescent assesses their achievements against their individual goals. This involves: 1) reading the number of steps recorded daily via the activity trackers . They signal the start of a specific activity, thus helping the transition between activities  to 2H. Aarphones .The research protocol and consent procedures were approved by The University of Sydney Human Research Ethics Committee (2017/272) and by the NSW Department of Education State Education Research Applications Process (2017151).The study was conducted in 2017 and 2018 with a sample of one control class (26 students) and 2 intervention classes (57 students) in schools located in the Sydney inner city area. To avoid information contamination, the study groups were conducted in two different schools and years. Written consent was obtained from the principals, class teachers, parents and students. All participants were consenting students attending a Year 5/6 class and were able to withdraw from the study at any time. The 2017 and 2018 studies were conducted over the same month (November) of each year and the data were combined for analysis. The program was delivered over 5 consecutive weeks. The research team attended the school to supervise the delivery of the program in the morning, usually between 9AM and 11AM. The control class received no program over the 5 weeks.The participants’ MVPA was objectively measured prior the program and after completion of the last iEngage module using a research grade accelerometer validated in young adolescents (GENEActiv) . The GEN© activity tracker continuously for the 5-week duration of the program on the non-dominant wrist. Misfit Ray© is a commercial tracker well tolerated by children and adolescents. Daily step counts were provided to the participant via the iEngage app during each module. In addition, raw data, averaged over 1 min periods were downloaded at the end of the program to allow analysis of daily steps consistency rate as previously described [For the iEngage classes participants wore a Misfit Rayescribed . Goals, Data analysis are presented for twenty-six control participants and fifty-seven iEngage participants. Seventy-one participants were included in the iEngage group. Fourteen were excluded from the analysis because they missed at least 3 modules due to absences, injury, or attendance to special study programs. No student dropped-out or asked to stop the program. Anthropometric, fitness and PA data from the activity tracker (daily steps collected at each module for 5 weeks) as well as GENEActiv data (collected and averaged over 3 school days before and after the iEngage program) are presented as mean +/- standard deviation (SD). Goals and achievements are presented as a percentage of students choosing or achieving a particular goal or PA level. To assess goal achievement for the whole cohort, data was aggregated over the 3 days preceding each module .There was no statistical difference between control and experimental groups at baseline for anthropometric data, physical activity or physical fitness . Most paP < .001, On average, participants progressively and significantly increased their daily steps goals from 6,153 ± 774 steps/day in M2 to 9,153 ± 1317 in M6 and finally 10,461 ± 760 in M10  = 91.1, P = 0.045, While participants could select 12,000 steps/day from module 8, 17% of participants set this goal in M8 compared to 27% in M10, with a marked difference between girls (7%) and boys (48%) . Across Within each module, students entered the daily steps recorded by the activity tracker for each of the previous three days. Results showed that students met or surpassed goals in the 3 days preceding modules M2, M3, M4, M6, M9 and M10 while they missed out on achieving goals in M5 and M8 . The proWe assessed participants’ satisfaction with the program with a simple question at the end of each module ("did you have fun in completing the module") using a Likert scale (1 to 5). The average score for the whole program was 4.3 ± 0.99.© activity trackers during the program showed that both female and male participants increased their daily steps over time (P < .001). Comparing the first 4 with the last 4 days of program showed that daily steps increased by 2,647 steps/d . Boys tended to increase MVPA more than girls  and after school, about one hour after school until 7PM . After sThis study is the first to report on the implementation and outcomes of a fully integrated mobile health program in primary school context, using both participant’s self-reported data and continuous monitoring of PA during the program. It shows that a digital health education and behaviour change program integrating activity tracker successfully guided adolescents to effectively progress PA goals and achievements. Data indicate that the program led adolescents to increase PA and MVPA in daily life through short bouts of activity during recess, lunch and after school. We show that the iEngage app can be used as a research platform to study how adolescents engage and respond to health education or behaviour change interventions.iEngage was based on BCTs, the Australian physical literacy framework and WHO recommendations for adolescents , 18, 29.Recent systematic reviews and meta-analysis found interventions using diverse degree of mobile health technologies targeting the promotion of healthy behaviours in schools, however none used mobile health , 32. NonMVPA can only be assessed through research grade accelerometers which do not provide information to the user on their PA. This was done before and after the program in the iEngage group and before and after a 5-week period in the control group over the same weeks of the school term. At baseline, daily MVPA in all participants was on average ~50 min /day with higher MVPA engagement in boys (~63 min) compared to girls (~43 min), and only 32% of the cohort achieving 60-min/day MVPA on average . Our datWhen analysing PA post- versus pre-intervention, previous systematic reviews and meta-analysis found no or little effect of school-based interventions, with no significant differences between those using or not web-based technology , 31. OurOne study  found thAdolescent boys and girls differed from each other with regards to both PA levels and the way they responded to the program. While all students developed competences in setting goals, girls tended to be less ambitious with setting daily step goals during the whole program, progressing goals slightly slower than boys. This indicates that, although they were exposed to the same program, the pace at which girls were willing to progress was slower than for the boys. This attitude toward goals setting cannot be explained by physical capacity since girls in the program had similar or slightly better performance across all baseline fitness tests. However, we cannot exclude the impact of perceived fitness or social norms on goal setting . While gWhile both girls and boys increased daily PA, boys were more successful than girls in increasing MVPA following iEngage. The greater enhancement of PA in boys compared to girls aligns with previous studies on school-based PA interventions including either single (PA) or combined (PA and nutrition) components  and with© activity tracker and to synchronise weekly with the app once the program is completed could encourage sustaining the behaviour over longer periods.One limitation of this study is the relatively small cohort however our results were consistent across the two schools. In each school, all year 5 and 6 students were included indicating that a whole class can participate in the program. iEngage is a short program with 10 modules delivered over 5 weeks. While this allows for strong focus and facilitates sustained motivation, a slower pace may be envisaged to enable progression and maintenance at slower rates, which may be more suitable for female adolescents. This study specifically focused on PA changes during the program and immediately after, however allowing the adolescents to keep the Misfit RayDelivered through a tailored digital environment, iEngage combines health education, experiential learning, interaction with peers and behavioural change theory. It enables individual goal setting, self-monitoring and self-assessment. In addition, iEngage provides researchers with a powerful tool to study how adolescents respond to information, activities and goals setting through continuous recording embedded in the app. In the future, iEngage could be considered to help adolescents maintain or increase PA in situations such as pandemic-induced lockdown and remote school activities but also in paediatric populations in which PA behaviour is important.