Cognitive and social capacities require postnatal experience, yet the pathways by which experience guides development are unknown. Here we show that the normal development of motor and nonmotor capacities requires cerebellar activity. Using chemogenetic perturbation of molecular layer interneurons to attenuate cerebellar output in mice, we found that activity of posterior regions in juvenile life modulates adult expression of eyeblink conditioning , reversal learning (lobule VI), persistive behavior and novelty-seeking (lobule VII), and social preference (crus I/II). Perturbation in adult life altered only a subset of phenotypes. Both adult and juvenile disruption left gait metrics largely unaffected. Contributions to phenotypes increased with the amount of lobule inactivated. Using an anterograde transsynaptic tracer, we found that posterior cerebellum made strong connections with prelimbic, orbitofrontal, and anterior cingulate cortex. These findings provide anatomical substrates for the clinical observation that cerebellar injury increases the risk of autism. Human capacities for cognition and flexible behavior unfold rapidly in the first six years of life. During this period, subcortical processing helps refine connections in the developing forebrain . Even thPediatric cerebellar insult causes cognitive and affective deficits . Indeed,The cerebellum’s role in guiding and shaping behavioral development is likely to be region-specific . AnatomiTogether, these previous findings suggest that the cerebellum plays a crucial role in the developmental maturation and adult expression of flexible and social behaviors. We tested this hypothesis using three tools. First, we used Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) to achieve reversible, anatomically-localized perturbation of a specific cell class in freely moving animals. DREADDs do not act unless exposed to an activator molecule . Using tTo probe the role of identified cerebellar regions  during cAs our developmental perturbation, after AAV injection at postnatal day (PND) 21 ; Table 1d = 1.5 pooled standard deviations for FF and 1.3 for CV2)  . Furtherfor CV2) .We tested whether DREADD activation could affect classical eyeblink conditioning, a cerebellum-dependent associative learning task . By pair-/-Tsc1 mice, which show cerebellar degeneration and ataxia . -/-Tsc1 mice showed considerably wider forelimb and hindlimb stance than controls , potentially arising from its conversion to a bioactive product  to obtain a panel of quantitative information . Acute CTo test flexible learning, we used a swimming Y-maze in which mice were habituated to a maze, initially taught to find an underwater platform in one maze arm, and later switched to the other arm . InitialFor all lobules, DREADD-injected mice treated in juvenile life with CNO learned at the same rate as uninjected mice . Acute ed = 1.1 for multisession reversal 1). Crus I developmental mice also showed decreased flexible behavior as measured by multisession reversal one and final reversal 1  . PCA ideThe first PC (‘PC1’), which by definition captured the most variance in the control group, was dominated by distance measures . In contTo probe social interaction, we monitored mice in a three-chambered apparatus  in which−6 to 0.006, paired t-test). Left/right chamber preference was uncorrelated with preference under baseline conditions , suggesting an absence of chamber bias arising from environmental factors such as odor or landmarks . There were no differences among control groups for any of the social chamber parameters (Disruption of crus I or crus II during juvenile life led to profound adult indifference between mouse and object as measured by time spent in the mouse chamber and in close proximity to the cup (rameters .d = 2.8 to 4.9), with no statistically detectable differences between them or with uninjected groups . Thus, the capacity of mice to express social preferences in the three-chamber apparatus did not depend acutely on activity in any cerebellar region tested, lobule VI or VII or crus I or II.Changes in preference for mouse over object were not seen in adult-injected mice for any lobule, 20 min after administration of CNO  for social chamber control groups was dominated by distance measures . In exped = 0.9 Šidák’s multiple comparisons post-hoc test p=0.02), as well as decreased exploration time  and distance . The effect on closed-arm preference was opposite to that seen in adult disruption, which enhanced open-arm preference , lobule VII for regulating persistive behavior  or novelty-seeking (three-chamber task), and crus I/II for the ability to express a social preference. These deficits set the stage to explore cerebellar endophenotypes using dimensionality-reduction and regression methods.In the past, the tasks of We surmised that cerebellar phenotype intensity might vary as a function of DREADD expression, consistent with the idea of the cerebellum as a feedback controller  and on tIn our linear models, the extent of DREADD expression in MLIs in each lobule was used as an input variable. To quantify expression, we reconstructed mCherry co-expression from two-photon tomographic images  or from For each behavioral metric, a linear model was fitted using four regressors, corresponding to the fraction of voxels containing label in each of the targeted lobules .Raw data for this study are available from the corresponding author upon request . Code and data is available at Experimental procedures were approved by the Princeton University Institutional Animal Care and Use Committee and performed in accordance with the animal welfare guidelines of the National Institutes of Health. Mice used in this study were C57BL/6J (referred to in the manuscript as ‘wild-type’ mice) males ordered through Jackson Laboratory . All mice had at least 48 hr of acclimation to the holding facility in the Princeton Neuroscience Institute vivarium before experimental procedures were performed. Mice in the acute cohort, four experimental groups for each lobule see , and ageL7Cre;Tsc1flox/flox male mutant mice  16 adult C57BL/6J male mice that received no treatment and no surgery were used as controls for Y-maze reversal, social chamber, grooming, gait and elevated plus-maze; (b) six adult C57BL/6J male mice that received saline injections but no surgery were used as controls for Y-maze reversal, social chamber, grooming, gait and elevated plus-maze; (c) eight adult C57BL/6J male mice that received GCaMP6f injections in lobule VI and CNO injections were used as sham controls for Y-maze reversal; (d) 16 adult C57BL/6J male mice that received headplate surgery were used as controls for eyeblink conditioning; (e) five adult C57BL/6J male mice that received DREADD injections into the eyeblink zone and saline throughout training served as sham controls for eyeblink conditioning. For eyeblink acute experiments we also injected 4 C57BL/6J male mice directly into a zone in paravermal lobule VI previously shown to directly drive the eyeblink conditioned response . To bencant mice  in whichAll mice were housed in Optimice cages  containing blended bedding , paper nesting strips, and one heat-dried virgin pulp cardboard hut . PicoLab Rodent Diet food pellets  and drinking water  were provided ad libitum. Mice were relocated to clean cages with new component materials every two weeks. All mice were group-housed in reverse light cycle to promote maximal performance during behavioral testing, which took time during the day.Surgeries on mice in the acute and developmental cohorts were performed in accordance with previously published procedures . In shorDesigner Receptor Exclusively Activated by Designer Drugs (DREADD) viral construct AAV8-hSyn-hM4D(Gi)-mCherry  was injected using borosilicate glass capillaries with an outer diameter of 1 mm and internal diameter of 0.58 mm . The pipettes were pulled using the Sutter Micropipette Puller  and bevelled at a 45 degree angle to a bubble number of ~5. Proper fluid flow through pipettes was verified before insertion into the brain. To ensure proper viral spread across lobules of interest, and to account for difference in sizes across lobules and ages during injection times in acute vs developmental group) between 250 and 900 nL of DREADD construct in total was injected per mouse, distributed across four specific locations see . SpecifiGeneral environment, transportation, and drug delivery. All behavioral tests in developmental and acute groups took place between PND 57 and PND 126. The order of behavioral testing was consistent for all groups: (1) social chamber, (2) grooming, (3) Y-maze reversal learning, (4) eyeblink conditioning, (5) elevated-plus maze, and (6) gait. After transportation from the holding facility, mice were acclimated to the behavioral testing room for at least 60 min before commencing a behavioral test. Behavioral testing took place between 9AM and 6PM under low white light illumination, red light (for elevated plus-maze), or darkness to reduce stress and not to disrupt the sleep-wake cycle of the tested mice. For the social chamber assay, novel mice were transported on a separate level of the rolling cart from the experimental mice and the cages were placed on separate levels in the behavioral testing room. Animals without a water bottle were given a 2-ounce HydroGel 98% sterile water gel  to prevent transient dehydration. In the acute experimental cohort, the DREADD agonist clozapine-N-oxide (CNO) was delivered through i.p. injection at a dose of 1 mg/kg 20 min before the beginning of behavioral testing. Developmental inactivation was achieved through oral CNO delivery in opaque water bottles at a dose of 10 mg/kg. In both cases, CNO was first dissolved in dimethyl sulfoxide which was then diluted to 1.5% concentration in sterile saline for i.p. injections or drinking water for oral administration. When indicated, mice received saline as a control for i.p. injections.Eyeblink conditioning. Eyeblink conditioning in all tested mice was performed between PND 84 and 126. Animals were trained as previously described  and two wooden blocks (56 cm x 5.5 cm) were positioned to create a walking path (50 cm x 6.5 cm) for gait analysis and this apparatus was elevated to a height of 56 cm above the ground. A lamp  illuminated the path from below. Mice were placed at the beginning of the path and gently encouraged to walk toward a cardboard box covering the terminus of the path. Between three and seven trials were conducted for each mouse. Trials were video-recorded from below with an iPhone6s  at 30 frames per second (1080 p) ((1080 p) . A ruler(1080 p) . Using aGrooming assay. Each mouse was transported to a clean, empty cage for the grooming assay. The cage rested on a wooden platform under a camera (PlayStation Eye) used to record the mouse during testing at 50 frames per second, using a custom-written Python 2.7.6 (Anaconda 1.8.0) script and CLEye Driver  was dissolved in the water to eliminate any platform visibility. At the end of the experimental day, all water was removed from the maze and the maze was cleaned with tap water, tissues, 70% ethanol and left to dry overnight. A black poster-board screen bordered three sides of the apparatus. A camera (PlayStation Eye) was mounted above the apparatus to record a field of view containing the entire apparatus at 50 frames/s, using a Python 2.7.6 (Anaconda 1.8.0) script and CLEye Driver (https://codelaboratories.com/products/eye/driver/). The mouse was lifted by the tail and inserted into the water 2 cm in front of the apparatus edge for each swimming trial. The tail was released in synchrony with initiating the video recording.Testing was performed for five consecutive days: Day 0 - Habituation; Day 1 - Acquisition; Day 2 - Test; Day 3 - Reversal; Day 4 - Reversal . HabituaThe Acquisition phase contained four sessions, each with five consecutive trials of 40 s duration each. The Y-maze apparatus contained a platform at the end of either the left or the right arm of the Y-maze. The arm with a platform was randomly chosen for each mouse. In the Test phase, each mouse experienced five trials with the platform in the same location as randomly determined in the Acquisition phase. Mice were required to have at least 80% success in their first choice turn-direction order to proceed with the behavioral assay. All animals included in the analysis passed this criteria and continued with the assay. In the Reversal phases, the location of the platform was the opposite of the trained location: mice trained in Acquisition and Test with the platform at the end of the right arm of the apparatus were introduced to a Y-maze apparatus with the platform at the end of the left arm, and vice versa. Each Reversal phase consisted of four separate sessions of five consecutive trials each of 40 s duration, followed by a fifth forced session in which a polycarbonate sheet was inserted into notched slots to prevent entry into the arm without the platform. After the conclusion of each swimming trial, the mouse was removed from the maze or platform and dried shortly with tissue before the start of the next trial. Between sessions, mice were placed in a clean cage with bedding under a heating lamp  until fully dry, then replaced in their original cages until beginning the next session. Mice were trained in groups of five to seven, thus maintaining an approximately equal amount of rest time between sessions for all mice. Performance metrics regarding the first-choice turn direction of the mouse in the Y-maze behavioral assay were determined through manual scoring and corroborated through an automated custom Python package. This analysis package tracked the position of the mouse during navigation of the Y-maze to determine first-choice turn direction for each trial as well as distance traveled and average velocity .Social chamber assay. The social chamber apparatus was custom-constructed according to established specifications (ications  as a traications  for traiications . BaselinElevated plus-maze assay. The polycarbonate elevated plus-maze (EPM) consisted of two shielded and two open arms  was used to track the centroid of the mouse throughout the ten minute session, with a user-drawn outline of the full arena as well as user-drawn rectangular outlines of both closed arms, both open arms, and the central crossroads area . This anTo capture this a variety of capacities, including anxiety and exploratory behaviors , we quanSocial chamber and Y-maze mouse tracking. Video was acquired at 50 Hz, 640 × 480 pixel resolution, using a Playstation Eye camera with the CLEye Driver. For each behavioral session, mouse tracking was performed using custom software written in Python  by the following procedure: a background image was computed as the gaussian-filtered mean projection of a 15 to 30 s period prior to insertion of the mouse into the apparatus. For each frame in the experimental session, the difference between the gaussian-filtered frame and the background image was computed. The difference image was thresholded according to a user-defined value, and edges of this binary image were detected using the Canny algorithm. Contours were extracted from the edge image, and the contour with the largest area was taken to represent the approximate outline of the animal. Contours were excluded if their center was greater than 100 pixels away from the location in the previous frame, if they were located outside the user-defined bounds of the apparatus, or if they were inside other user-defined areas containing features that interfered with tracking accuracy. For frames in which the algorithm found no suitable contour, the position of the mouse was inferred to be in its most recent location. For the social chamber the boundaries of the three chambers were demarcated manually and the time spent in each chamber was determined from the number of frames in which the animal was detected in each chamber. For Y-maze, the location of the mouse in each frame was automatically assigned to a specific sub-portion of the maze using fiducial markers on the maze apparatus. Code for tracking is available from the corresponding author upon request.After successful completion of all behavioral assays, experimental mice were anesthetized with an overdose of ketamine (400 mg/kg)/xylazine (50 mg/kg) (i.p.) and perfused with 4% paraformaldehyde (PFA). Brains were stored in 4% PFA for three to six hours at room temperature followed by overnight incubation in 4°C. After rinsing with phosphate buffered saline (PBS) the brains were placed in PBS with 0.1% sodium azide and shipped to TissueVision  for processing via serial two-photon tomography imaging. Endogenous tissue autofluorescence (500 – 540 nm) and mCherry DREADD signal (600 – 680 nm) were imaged in coronal slices at intervals of 50 μm. Images received from TissueVision were registered to the Allen Brain Mouse 25 μm slice Common Coordinate Framework Reference Atlas Version 3 ; see ‘QuMice used for characterization of the DREADD expression pattern were anesthetized with an overdose of ketamine (400 mg/kg)/xylazine (50 mg/kg) (i.p.) and perfused with 4% PFA 4 to 5 weeks following the injection -mCherry virus; viral injections and surgeries were performed as described above). Following 2 hr post-fixation at room temperature, cerebella were isolated from the rest of the brain, incubated overnight at 4°C in 10% sucrose and embedded in gelatin (12% gelatin/10% sucrose). Gelatin embedded brains were then hardened for 1.5 hr at room temperature in 30% sucrose/10% formaldehyde, incubated overnight at 4°C in 30% sucrose, rapidly frozen, sectioned sagittally at 50 μm and collected in 0.1 M PBS. Sections were processed for immunohistology by washing with PBS and counterstained with DAPI for 10 min (30 μl/ml) and mounted on glass slides with Vectashield anti-fade mounting medium . Brains from four mice used for in vivo electrophysiology were isolated and processed in the exact same way.Brains from mice injected with the herpes simplex virus (HSV) one strain H129 causing neuronal expression of EGFP  were first anesthetized with isoflurane in their home cages before being injected with ketamine (400 mg/kg)/xylazine (50 mg/kg) (i.p.) and perfused under the animal biosafety level 2 conditions with 4% paraformaldehyde with heparin (20 units/ml) were isolated, postfixed, incubated and prepared for sectioning as described above. Whole brain sagittal sections were cut at 50 μm and collected in 0.1 M PBS. Sections were processed for immunohistology by washing with PBS and incubating for 1 hr at room temperature in a blocking buffer  prior to a 3-day incubation at 4°C in PBS buffer containing 2% NGS, 0.4% Triton and the chicken anti-GFP primary antibody -mCherry virus used for in vitro electrophysiological experiments were transferred from the artificial CSF (aCSF) to 4% paraformaldehyde and stored overnight at 4% paraformaldehyde. Fixed slices were then washed with PBS and mounted on glass slides with Vectashield.The majority of brains were imaged via serial two-photon tomography by the TissueVision company. A smaller subset of brains were cut with a cryostat and imaged at 10x magnification on an epifluorescence Nikon Eclipse Ti microscope  or on an epifluorescence Zeiss Axioplan2 upright fluorescence microscope . In order to obtain high-resolution images, selected sections were scanned with a Leica SP8 confocal laser-scanning microscope  using 10x, 40x or 63x objectives, hybrid (HyD) detectors for sensitive detection, and sequential scan mode. Images used in In order to standardize histological expression analysis across animals, we devised a pipeline for sample registration to the Allen Brain Atlas . We opteFirst, two-dimensional boundaries of anatomical regions of interest within the cerebellum were manually traced within each section for each sample as well as the reference volume  using Neurolucida software . Additional tracing was performed to outline the injection volume within the same images for the sample brains. Once traced, contours were extracted as a set of line segments defining closed polygons for each connected component within a section. The interior of section-wise polygons were binarized and the volume was resampled to yield an isotropic volume for each structure. Binary volumes were then converted to triangular surface meshes via isocontour thresholding at isovalues of 1, generating a set of vertices and edges reflecting the underlying closed volume to be used for registration.In order to use the shape and location of brain structures that were clearly visible as landmarks for aligning the sample to the reference volume we devised a point-based registration procedure. For a given pair of surface meshes, we first applied a rigid registration between the two sets of vertices to approximately account for translation and orientation differences. Then, this registration was refined through a consensus-based nonlinear point set registration algorithm . As the For joint behavioral-anatomical analysis, viral expression volumes were max projected across the anterior-posterior axis to yield mediolateral/dorsoventral projections, and then further reduced to vectors by taking the average number of expression signal voxels along each coordinate of the mediolateral axis. In ‘masked’ analyses, the ROI under consideration was used as a voxelwise inclusion filter before reducing sample volumes to expression vectors. All correlations were computed using Spearman’s ρ and p-values using exact permutation distributions as implemented in MATLAB’s corr function (MathWorks Inc.).For the linear model analysis, models were fit relating anatomical expression to each behavioral metric with continuous values . Given that the control groups had no expression only experimental (injected) groups were used. Therefore, we regressed the behavioral scores of adult and juvenile experimental cohorts with DREADD injections, onto their respective expression profiles. One model was fit for each behavioral metric, with the value of that metric for each mouse as the predicted variable. Inputs to the model consisted of four values for each mouse: the fraction of lobule containing label for each of lobule VI, lobule VII, crus I, and crus II. Coefficients were fit for each of these terms along with an intercept term. The fraction of expressing voxels for each mouse is shown in As a means of describing the natural underlying structure of the behavioral phenotypes measured through the assay-specific metrics, we performed principal component analysis (PCA) on the set of observations in control mice to define a linear subspace of correlated behavioral measures. The resulting principal components or ‘eigenbehaviors’  were used as a basis onto which both control and experimental mouse data were projected after normalization by centering to the mean and standard deviation of the controls. Both principal component scores and normalized raw metrics . 250 μm sagittal slices of the vermis were cut on a vibratome . Slices were incubated in slicing medium at near physiological temperature (34°C) for 2 mins and transferred to 34°C aCSF for 30 min. Slices were then held at room temperature in a chamber filled with oxygenated aCSF covered with aluminum foil to prevent too much light exposure and used within 6 hr. Experiments were performed in aCSF at near physiological temperature ~33°C. The patch pipettes (6 – 8 MΩ) were filled with intracellular solution containing (in mM): 120 potassium gluconate, 9 KCl, 10 KOH, 3.48 MgCl2, 4 NaCl, 10 HEPES, 4 Na2ATP, 0.4 Na3GTP, 17.5 sucrose and 10 μm Alexa 488 (at pH 7.25). CNO (10 μM in aCSF) was applied to the slices using positive pressure via another patch pipette (>2 MΩ) positioned above the recording area. Data was stored and collected for offline analysis using the MiniAnalysis program .Two male, 6-week-old C5B7L/J6 mice were anesthetized with isoflurane and injected in lobule VIb with AAV8-hSyn-hM4D(Gi)-mCherry virus as described in ‘Animal Preparation’. Two to 5 weeks later, cerebella were removed and dissected in ice-cold oxygenated slicing medium containing (in mM): 93 N-methyl-D-glucamine HCl, 2.5 KCl, five sodium ascorbate, two thiourea, three sodium pyruvate, 0.5 CaClwww.neurasmus.com), running under MATLAB .Four male, 6-week-old C5B7L/J6 mice were anesthetized with isoflurane and injected in lobule VI with AAV8-hSyn-hM4D(Gi)-mCherry virus as described in the ‘Animal Preparation’ section. Following the injection mice were fitted with a cranial 3-mm-wide window covered by a removable silicone plug  and a custom-made two-piece headplate . Two weeThe HSV1 strain H129, which contains the EGFP transgene, was used for multisynaptic anterograde tracing  test. Inclusion of the non-parametric KS enabled identification of potential differences in the overall distribution of behaviors in addition to distribution mean and median. 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, including Jennifer Raymond as the Reviewing Editor and Reviewer #3, and the evaluation has been overseen Richard Ivry as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Jörn Diedrichsen (Reviewer #2).Thank you for submitting your article "Normal cognitive and social development require posterior cerebellar activity" 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.Summary:This manuscript addressed an important topic in neuroscience, the role of the cerebellum in normal cognitive and social development. More specifically, this manuscript presents evidence that transient perturbation of cerebellar activity during postnatal development can have long-term effects on cognitive function. A chemogenetic approach was used to selectively perturb activity in four different cerebellar regions in juvenile mice, followed by behavioral testing in adult mice; and the results are compared with the effects of acute chemogenetic inactivation in adult mice during behavioral testing. This experimental study design is powerful. Although the cerebellum has been previously implicated in cognitive functions, and developmental perturbations have been used previously, the unique contribution of the present study design is the ability to compare transient inactivation of the cerebellum in juveniles versus adults. The results are likely to have significant scientific and translational impact. However, the presentation of the results could be significantly improved to make the manuscript clearer and stronger.Essential revisions:1) The core Results sections  are complicated, and it is difficult to get a full overview of the results. Also, the authors should clarify which results are "punch line" results.The PCA analysis and linear regression analysis only help here partially.One possibility would be (a) z-standardize all behavioral scores of all groups and all task relative to the control group, with 0 meaning unimpaired (b) present a figure similar to the one in Figure 3E, but including both acute and developmental groups and the non-motor and motor test (locomotion and eye blink conditioning). This table will allow the reader to visually get a quick overview over the results – especially if you mark significant deficits.eLife), with all of the relevant controls, most importantly, the direct comparison of adult vs juvenile inactivation of a given cerebellar region with appropriate statistical comparisons of the two (see point #2).In addition, each of the behavioral tasks is worthy of its own main figure or two (there is no limit on number of figures at 2) In its present form, the presentation of the results does not leverage the power of the experimental design. The main interesting contrast – the one between developmental and adult perturbation – is strangely under-emphasized. The results should be presented to facilitate and focus on the direct comparison of juvenile vs adult inactivation of the same cerebellar region on each of the behavioral tasks.The direct developmental-acute contrast is reported only sometimes, and it is very difficult for the reader to get a full impression of similarities and differences. The reader should not have to go back and forth between main figures and supplementary to try to cobble together this key comparison. Moreover, after investing the effort to do so, it was in some cases still difficult to know what to conclude. For example, the controls' performance on open arm preference in Figure 2I va Figure 2—figure supplement 2C varies by almost twofold, which is similar to the effect sizes for cerebellar inactivation, which the authors highlight. This makes one wonder how to interpret the results. Does the difference between the two control groups arise from testing at different ages? If so, then this needs to be addressed in comparing the results of juvenile vs acute cerebellar inactivation.Some analysis of how equivalent the juvenile and adult manipulations are in terms of their localization and amplitude is needed. Since the injections of virus were done at different ages, it would be helpful for the authors to provide a direct comparison of where the DREADDs are expressed after the juvenile vs adult injections, including an analysis of which cell types express the DREADs. This is especially important since the effect of adult acute inactivation of lobule VII  looks very much like the effects of juvenile inactivation of Crus 1 , and the histology shown in Figure 3D suggests that those two types of injections are, in aggregate, overlapping.A full overview over the results (see comment #1) will help here – but as an additional analysis you could test how similar the full functional profiles  are between each developmental and control mouse (as compared to the within-group correlations). Either way, a clear discussion of the possible lasting cortical or cerebellar changes caused by a developmental perturbation is needed.3) The principle component analysis and its conclusions are not very clear. The main problem with the current analysis is that it is performed for each task separately and does not include the motor measures and therefore fails to give a global overview of the data. Secondly, the author seems to rely on the hope that the principle component analysis will somehow find useful and semantically meaningful directions in the behavioral profile from the variation in the control mice. It is not clear that this hope is substantiated. If one of the PCs reflects motor behaviors only, should it not be impaired clearly by one of the lesions? The authors should consider removing this analysis in favor of a careful visual display of the global results, as suggested above. The PCA analysis can still be used for visualization as in the scatterplots in Figure 3B.4) The negative weights in the linear regression analysis are puzzling. Do these mean that the experimental group performed better than the control? This depends on a number of things: Did you include the values for the control group into the regression? Did you include an intercept? Did you standardize scores before? Should the linear regression weights not show a very similar pattern from the significant group effects – it seems from Figure 1E that only crus I and the social chamber tasks are significant? There are currently simply not enough details given about the analysis to make sense of the results.5) Throughout the text you seem to draw analogies between "homologous" regions in the mouse and human – based on lobular annotation. Looking at what we know about the lobular connectivity patterns in both species, the homologies (which are tricky to establish anyway) cannot necessarily be established on a lobules-by-lobules basis. Also, the connectivity pattern in humans does not really respect lobular boundaries if you inspect the Buckner et al., (2011) results carefully. Therefore, it is very likely that connectivity will have shifted substantially in evolution – and your tracing results (while showing specificity) do seem to confirm this. The current study provides absolutely valuable insights into the role of cerebellar circuits in development and adulthood – but mouse-human analogies should be discussed with more care.6) The generalization from specific measurements on specific behavioral tasks to broad cognitive categories like novelty seeking and sociability is excessive. A subtle effect on one behavioral assay does not necessarily mean that the effect is truly the result of the label the investigators put on that assay e.g., a social deficit vs some other specific feature of the task. The results of the sociability test in Figure 2F suggest that the biggest effect is not a decrease in time with the other mouse, but rather an increase in time with the object, which one might equally be labelled a broadening of curiosity rather than a decrease in sociability. There are many additional places in the manuscript where the labeling of specific measurements on specific assays seemed too loose, and too contrived to make a case about autism . Fine to do some speculation in the Discussion section, but more restraint and rigor in the interpretation would strengthen the manuscript.eLife. Your article has been reviewed by three peer reviewers, including Jennifer L Raymond as the Reviewing Editor and Reviewer #3, and the evaluation has been overseen Richard Ivry, the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Akira Sawa (Reviewer #1); Jörn Diedrichsen (Reviewer #2).Thank you for submitting your article "Normal cognitive and social development require posterior cerebellar activity" 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.Summary:This manuscript addressed an important topic in neuroscience, the role of the cerebellum in normal cognitive and social development. More specifically, this manuscript presents evidence that transient perturbation of cerebellar activity during postnatal development can have long-term effects on cognitive function. A chemogenetic approach was used to selectively perturb activity in four different cerebellar regions in juvenile mice, followed by behavioral testing in adult mice; and the results are compared with the effects of acute chemogenetic inactivation in adult mice during behavioral testing. This experimental study design is powerful. Although the cerebellum has been previously implicated in cognitive functions, and developmental perturbations have been used previously, the unique contribution of the present study design is the ability to compare transient inactivation of the cerebellum in juveniles versus adults.In response to the previous review, the authors have significantly reorganized the presentation of the results in a way that increases clarity tremendously. The experiments are well controlled and rigorously analyzed. The systematic comparison of {different behavioral tasks and behavioral metrics} X {developmental time point for the perturbation} X {regional variation} provides a rich and valuable data set. The finding of different effects of developmental vs adult perturbations is novel and should be of broad interest. The reviewers have only two remaining concerns.Essential revisions:1) In response to the reviewers' previous query about negative weights in the linear regression, the authors now use absolute value. This is a step in the wrong direction, as it obscures whether more lesion in the structure improved or impaired function. A better approach may be to show the signed value, but flip the sign, so that positive values consistently  indicate impairments and negative values improvement in function (or the other way around). Again, removing the sign and therefore this crucial information did not address the reviewers' concern.Related to this, the questions about inclusion of control group and intercept were not answered clearly. One might guess that the control group was not included, but all 4 injection groups, separately for the adult and juvenile cohorts? Please clarify. We thank the reviewers for their careful attention to our manuscript. Presenting a large and multidimensional body of experimental work is a challenge. The reviewers’ suggested revisions are a substantial improvement.We have edited the manuscript and reorganized the figures to address the points raised by the reviewers. We now present acute and developmental cerebellar perturbations side by side. We also discuss all differences in the behavioral tests instead of highlighting just a few. Finally, we include a table where we summarize the resultsOur point-by-point response to comments is given below.Essential revisions:1) The core Results sections  are complicated, and it is difficult to get a full overview of the results. Also, the authors should clarify which results are "punch line" results.The PCA analysis and linear regression analysis only help here partially.One possibility would be (a) z-standardize all behavioral scores of all groups and all task relative to the control group, with 0 meaning unimpaired (b) present a figure similar to the one in Figure 3E, but including both acute and developmental groups and the non-motor and motor test (locomotion and eye blink conditioning). This table will allow the reader to visually get a quick overview over the results – especially if you mark significant deficits.We now summarize principal results in units of effect size in Table 3.In addition, each of the behavioral tasks is worthy of its own main figure or two (there is no limit on number of figures at eLife), with all of the relevant controls, most importantly, the direct comparison of adult vs juvenile inactivation of a given cerebellar region with appropriate statistical comparisons of the two (see point #2).We have now rearranged the figures in this manner.2) In its present form, the presentation of the results does not leverage the power of the experimental design. The main interesting contrast – the one between developmental and adult perturbation – is strangely under-emphasized. The results should be presented to facilitate and focus on the direct comparison of juvenile vs adult inactivation of the same cerebellar region on each of the behavioral tasks.The direct developmental-acute contrast is reported only sometimes, and it is very difficult for the reader to get a full impression of similarities and differences. The reader should not have to go back and forth between main figures and supplementary to try to cobble together this key comparison. Moreover, after investing the effort to do so, it was in some cases still difficult to know what to conclude. For example, the controls' performance on open arm preference in Figure 2I va Figure 2—figure supplement 2C varies by almost twofold, which is similar to the effect sizes for cerebellar inactivation, which the authors highlight. This makes one wonder how to interpret the results. Does the difference between the two control groups arise from testing at different ages? If so, then this needs to be addressed in comparing the results of juvenile vs acute cerebellar inactivation.We now place adult/developmental data together in the same figure, and directly next to one another as a matched pair as appropriate.Some analysis of how equivalent the juvenile and adult manipulations are in terms of their localization and amplitude is needed. Since the injections of virus were done at different ages, it would be helpful for the authors to provide a direct comparison of where the DREADDs are expressed after the juvenile vs adult injections, including an analysis of which cell types express the DREADs. This is especially important since the effect of adult acute inactivation of lobule VII  looks very much like the effects of juvenile inactivation of Crus 1 , and the histology shown in Figure 3D suggests that those two types of injections are, in aggregate, overlapping.In all cases, expression was confined to molecular layer interneurons . We have added a mention of this to the abstract.We fully agree that it is necessary to address the fact that injection regions overlap. This was the rationale for the linear model. We now mention the linear model in the Abstract and explain it at greater length in the Results section.A full overview over the results (see comment #1) will help here – but as an additional analysis you could test how similar the full functional profiles  are between each developmental and control mouse (as compared to the within-group correlations). Either way, a clear discussion of the possible lasting cortical or cerebellar changes caused by a developmental perturbation is needed.In response to this comment, we attempted to visualize the mouse-to-mouse variability using multidimensional scaling. However, the results did not grab the eye. One likely reason is the variability of DREADD expression.In our view, this point is made most powerfully by the linear model. We have expanded that figure to include example scatterplots of the relationships that go into the linear model. These capture both, the variability and the nature of the functional contribution of each lobule.3) The principle component analysis and its conclusions are not very clear. The main problem with the current analysis is that it is performed for each task separately and does not include the motor measures and therefore fails to give a global overview of the data. Secondly, the author seems to rely on the hope that the principle component analysis will somehow find useful and semantically meaningful directions in the behavioral profile from the variation in the control mice. It is not clear that this hope is substantiated. If one of the PCs reflects motor behaviors only, should it not be impaired clearly by one of the lesions? The authors should consider removing this analysis in favor of a careful visual display of the global results, as suggested above. The PCA analysis can still be used for visualization as in the scatterplots in Figure 3B.For simpler presentation, we now use within-group variance of single metrics. We have changed the figures to focus on this rather than PCA.It is true that none of our lesions impaired PC1, which captures the largest share of behavioral variation in control groups. This is in fact the notable observation: PC1 does not capture the variability within a treatment group. In our view this is a rigorous way to test the idea that perturbations do something unusual to mice.4) The negative weights in the linear regression analysis are puzzling. Do these mean that the experimental group performed better than the control? This depends on a number of things: Did you include the values for the control group into the regression? Did you include an intercept? Did you standardize scores before? Should the linear regression weights not show a very similar pattern from the significant group effects – it seems from Figure 1E that only crus I and the social chamber tasks are significant? There are currently simply not enough details given about the analysis to make sense of the results.Negative coefficients mean that more expression is associated with a lower score on that metric. If positive values indicate more function, a negative coefficient means there is impairment. To make things clearer, we now display absolute value. The direction of change is shown elsewhere.5) Throughout the text you seem to draw analogies between "homologous" regions in the mouse and human – based on lobular annotation. Looking at what we know about the lobular connectivity patterns in both species, the homologies (which are tricky to establish anyway) cannot necessarily be established on a lobules-by-lobules basis. Also, the connectivity pattern in humans does not really respect lobular boundaries if you inspect the Buckner et al., (2011) results carefully. Therefore, it is very likely that connectivity will have shifted substantially in evolution – and your tracing results (while showing specificity) do seem to confirm this. The current study provides absolutely valuable insights into the role of cerebellar circuits in development and adulthood – but mouse-human analogies should be discussed with more care.In the Introduction we now emphasize that the anteroposterior lobular boundaries are useful as anatomical landmarks, but not necessarily functional boundaries. We now clarify that microzones are the established organizing principle of cerebellar cortex.6) The generalization from specific measurements on specific behavioral tasks to broad cognitive categories like novelty seeking and sociability is excessive. A subtle effect on one behavioral assay does not necessarily mean that the effect is truly the result of the label the investigators put on that assay e.g., a social deficit vs some other specific feature of the task. The results of the sociability test in Figure 2F suggest that the biggest effect is not a decrease in time with the other mouse, but rather an increase in time with the object, which one might equally be labelled a broadening of curiosity rather than a decrease in sociability. There are many additional places in the manuscript where the labeling of specific measurements on specific assays seemed too loose, and too contrived to make a case about autism . Fine to do some speculation in the Discussion section, but more restraint and rigor in the interpretation would strengthen the manuscript.We now specify parameter name in the social assay to clarify what measured quantity actually changed. We save our most speculative thoughts for the Discussion section.Essential revisions:1) In response to the reviewers' previous query about negative weights in the linear regression, the authors now use absolute value. This is a step in the wrong direction, as it obscures whether more lesion in the structure improved or impaired function. A better approach may be to show the signed value, but flip the sign, so that positive values consistently  indicate impairments and negative values improvement in function (or the other way around). Again, removing the sign and therefore this crucial information did not address the reviewers' concern.We have restored the signed information, and redefined the sign of each behavioral measure so that lost function is indicated as a positive value. We now define the sign conventions for impairments to Table 2. This information is reflected in Table 3 (and its supplement) and Figure 10.Related to this, the questions about inclusion of control group and intercept were not answered clearly. One might guess that the control group was not included, but all 4 injection groups, separately for the adult and juvenile cohorts? Please clarify.The linear regression model that we are presenting correlates the phenotypic scores with the amount of expression. Given that the control groups have no expression only experimental (injected) groups were used. Thus we regressed the behavioral scores of 4 injection groups, separately for the adult and juvenile cohorts, onto their respective expression profiles. An intercept was included in the fit. The fraction of voxels that had expression can be seen for each mouse in Figure 10—figure supplement 1. We have clarified that in the Results section and in the Materials and methods section.We have restored the signs of the regression weights as requested. In addition, we colored Table 3 (and its supplement) and Figure 10 so that red indicates impairment in function and blue an improvement.


Calcium dynamics in presynaptic terminals regulate the response dynamics of most central excitatory synapses. However, this dogma has been challenged by the hypothesis that mobility of the postsynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid subtype glutamate receptors (AMPAR) plays a role in tuning fast excitatory synaptic transmission. In this review, we reevaluate the factors regulating postsynaptic AMPAR mobility, reassess the modeling parameters, analyze the experimental tools, and end by providing alternative ideas stemming from recent results. In particular, newer methods of labeling AMPARs with small fluorophores in live neurons, combined with super-resolution microscopy and sub-second dynamics, lends support to the idea that AMPARs are primarily within the synapse, are greatly constrained, and have much slower mobility than previously thought. We discuss new experiments which may be necessary to readdress the role of postsynaptic AMPAR mobility in tuning fast excitatory synaptic transmission. The influx of Ca2+ then triggers the fusion of a primed glutamate-containing presynaptic vesicle. Results from modeling work indicate that once a vesicle fuses with the plasma membrane, glutamate concentrations quickly increase at the synaptic cleft, reaching an estimated 1-3 mM within 50 microseconds , where it then activates postsynaptically opposed glutamatergic receptors such as AMPARs. The activation of AMPARs is spatially restricted to a 125 nm radius from the center of glutamate release. This dynamic processes results in activation of only a fraction of all postsynaptic AMPARs . The replacement of the desensitized postsynaptic AMPARs allows the synapse to respond to the second incoming stimulus and activate another pulse, generally somewhat smaller than the first, although occasionally equal or larger to it. Their hypothesis also supposes that the exchange of desensitized AMPARs via diffusion is faster than the rate of recovery of AMPARs from the desensitize state (stimulation and trigger postsynaptic AMPAR desensitization . At most factors . Findingze state . The souze state .This revolution in thinking led by Choquet and colleagues about excitatory glutamatergic synapses has been possible with the advent of high-resolution single-particle microscopy techniques and electron microscopy (EM). These techniques have shown that surface-expressed AMPARs diffuse at synaptic and extrasynaptic areas of dendrites . The recFigure 1B). At larger spines, AMPARs cluster into nanodomains which are served by a glutamate activation zone of about 250 nm in diameter  and are then replaced by diffusion of naïve AMPARs (green dots) from adjacent nanodomains . This idea of receptor exchange between intrasynaptic nanodomains is herein referred to as the nanodomain hypothesis. This challenged the longstanding belief that presynaptic mechanisms solely underlie the paired-pulse response.The nanodomain organization of synaptic AMPARs forms the basis for a newer model of how postsynaptic AMPAR mobility plays a role in recovering desensitized AMPARs during paired-pulse stimulation . In thisdiameter . Becausediameter , CompansIn this review we will revisit the nanodomain hypothesis and the factors regulating postsynaptic AMPAR mobility and diffusion within nanodomains. We will particularly focus on understanding the dynamics of AMPAR exchange between intrasynaptic nanodomains and the role of AMPAR mobility in the paired-pulse synaptic response. We conclude that recent measurements, most notably super-resolution microscopy with small fluorescent labels, show that AMPARs do not have a enough freedom to move around the synapse, and hence, the nanodomain hypothesis, by itself, fails to explain the paired-pulse response. We present some other hypotheses which might help explain the observed changes in paired-pulse response.2] than larger ones. Spines with small postsynaptic membranes  have a lower number of postsynaptic AMPARs than larger mushroom spines , glutamate will quickly diffuse through the whole synaptic cleft and extrasynaptic membrane. This is expected to gate all postsynaptic AMPARs ; a fraction of those will fully gate and others will desensitize without opening  of 0.1 μm2/s   . This suigure 2) .Figure 2 shows examples of those images for a very small synapse (top row) and a large synapse (lower row). The large synapse shows that about 50% of the postsynaptic membrane is filled with small membrane-bound particles (quantified here by Image J). There are over 250 particles in this spine, including 79 particles labeled with an anti-GluR1 (now GluA1) antibody and about 30 labeled by an anti-NR2B (now GluN2B) antibody . The rest of these membrane-bound particles may belong to either mGluR1 and 5, Ca2+ and K+ channels, neuromodulatory receptors, transsynaptic proteins, or other ionotropic channels , a measure of average mobility, was 0.02 μm2/s for extrasynaptic particles, 0.0006 μm2/s for synaptic particles, and even slower for particles in zones heavily enriched with PSD-95. This constrained diffusion in the PSD is expected since the PDZ ligand domains of TARPS are responsible for trapping and immobilizing stargazin, and the associated AMPARs, at synapses . The reason for this is unknown but it may be because the numbers of both AMPARs and scaffold proteins increases linearly with the size of the postsynaptic spine. One caveat is that the smaller spines analyzed in this paper are already large in size in relation to what is considered a small spine (spine sizes < 0.05 μm2); thus the effects of molecular crowding may be different for very small stubby spines with relatively low particle density. Taken together, these recent studies have shown that molecular crowding at large spines constrains the speed, range, and directionality of movement for intrasynaptic AMPARs.In addition to the immobilization of surface-expressed AMPARs by binding to the MAGUKs, inst for AMPARs depends on the developmental age of the neurons  to 0.0075 μm2 (within a 86.6 nm circle). In these experiments they also observed a slight shift toward greater mobility with a low concentration of glutamate. However, most importantly, during realistic conditions of glutamate release such as following glutamate uncaging, the authors observed very little change in mobility. Under these conditions, no alteration in AMPAR mobility was observed in the first time point of the mean-square displacement (MSD) curve (50 ms time step)∗ as seen in Figure 6C of inst after glutamate stimulation was reported to be about 0.01 μm2/s, or tenfold lower than the required Dinst of 0.1 μm2/s. In addition, the non-stimulated spines showed a reduction in mobility. Therefore, it is still unclear how AMPAR/TARP dissociation will impact synaptic transmission during paired-pulse depression.The low number of fast moving AMPARs suggests that if AMPAR mobility plays a role during paired-pulse depression, some additional mechanism should be at play. Taken together, the evidence suggests that the high packing density of the PSD and the large number of surface expressed proteins impose a great restriction to the mobility of synaptic AMPARs. Accordingly, recent data shows that the majority of AMPARs (∼86%) do not move more than 25 nm in 50 ms which supports the view that the PSD is an environment of slow diffusion .∗These are the values extracted from the MSD, or abscissa, for all two consecutive time points of all particles. The Dinst is obtained from the linear fit to the first 3–4 points of the MSD versus time curve for each individually tracked particle . The MSD curve is calculated by squaring the mean distance for the duration of a time step according to the MSD formula:n (0) is the reference position for each individual particle, Xn(t) is the particle position determined at that moment in time t. In most experiments, the precision accuracy of drift-corrected recordings is below the total distance traveled by the AMPARs in one time step or 30 nm. Average single time steps or average Dinst should accurately describe differences in behavior of these particles.Where N is the number of particles that are averaged, Xinst of 0.1 μm2/s  the technical limitations of using bQDs to extract the values of Dinst from the MSD curve. In the experiments using bQDs, over 60% of particles move with Dinst larger than 0.1 μm2/s. As a result, many modeling papers have used this value as the mean Dinst value , about 75% of surface-expressed AMPARs move slower than 0.01 μm2/s or move about 22 nm, 96% of surface AMPARs move slower than 0.1 μm2/s or move about 50 nm in 50 ms, and only the remaining 4% of surface AMPARs move slower than 0.5 μm2/s, or about 158 nm in 50 ms.  than receptors tagged with the mEOS3.2-GluA2  the use of an artefactually high value of instantaneous diffusion coefficient of Dst value . However.2-GluA2 ; althoug.2-GluA2 . Therefoinst, which off-hand, might seem counter-intuitive. The reason is that with smaller probes, the AMPAR are more likely to be labeled inside of the synapse, where AMPAR movements are more constrained. For example, Lee and colleagues noticed that when AMPARs are labeled with fluorophores of smaller sizes, they are primarily in the synapse—75% of AMPARs are synaptic when labeled with small quantum dots (sQDs) and 90% of AMPARs are synaptic when labeled with the small CF633- or Atto647N-tagged streptavidin. However, only 5% of AMPAR is synaptic when labeled with big QDs. As seen by others  that the bQDs have a hard time accessing the synaptic cleft. However, when Lee and colleagues imaged neurons labeled with small QDs, the small QDs remained inside the postsynaptic spines for at least 15 min; supporting the idea that small QDs have unobstructed access to the synaptic cleft and synaptic AMPARs have an extremely long residency time.We argue that smaller probes give slower values of Dy others , the labrs  the bQDs might dislodge the AMPARs from the PSD, (2) bQDs may not be able to penetrate the synaptic cleft well enough to become anchored by the scaffolding proteins, or (3) bQDs may dissociate the AMPARs from the auxiliary TARPS. The smaller fluorescent probes do not appear to have this problem, probably because they can easily penetrate the synaptic cleft and are less likely to sterically interfere with the stability of the synaptic AMPARs. More reassuring is the fact that all values of Dinst obtained using the smaller labels—whether the small QDs (<12 nm diameter) or the streptavidin-organic fluorophores (<5 nm diameter)—are similar. Thus, we believe that the values of Dinst obtained using big commercial QDs are artefactual in nature while the values obtained using the small labeling tags most closely track the true mobility of endogenous synaptic AMPARs  in the synaptic cleft.  The minimal unit of a crosslinking complex is achieved when at least two AMPARs are recognized by either one primary antibody or by two primary antibodies tethered together by a secondary antibody. The number of antibodies present in the crosslinking complex depends on: (1) the abundance of epitopes, (2) the access of the antibody to the AMPAR, and (3) on how much primary and secondary antibodies are applied to the sample. If high concentrations of primary antibodies are applied to saturate all epitopes, then more than two AMPARs are expected to be crosslinked. In a hypothetical scenario that three AMPARs are crosslinked, this crosslinked complex will contain 3 AMPARs, between 2 and 4 primary antibodies, and between 2 and 4 secondary antibodies. In terms of size limitations, the linear size of the crosslinking complex is at least over 24 nm is size when bound to the N-terminus domain of the AMPAR. In terms of the width of these complexes, the crosslinking antibodies could form a large macromolecular complex that may surpass several million Dalton in size and exceed the size of the big QDs in hydrodynamic diameter.  have been studied in In Chamma’s study, they specifically addressed the effect of different-sized tags on the ability to provide good synaptic labeling of neuroligin in live labeling experiments on cultured neurons. In general terms they observed that smaller-sized tags yielded better synaptic labeling than larger size tags. The authors scored localization of the labeled neuroligin in relation to the center of the Homer labeling (used as a marker of the PSD) as a proxy for the penetration of the extracellularly applied labeling molecule. Good penetration was scored as perfect axial alignment with the Homer labeling; partial penetration was scored as partial overlay. They used labels of different sizes: the small monomeric streptavidin (mSA 12.5 KDa or less than 4 nm in size), tetrameric streptavidin (SA 52.8 KDa and slightly over 5 nm in size), and a full-sized primary antibody against neuroligin  all labeled with Alexa647. They found that the full-sized antibodies and tetrameric SA stained neuroligin outside of the Homer label, while the small monomeric streptavidin stained neuroligin right in the center of the Homer label. This suggests that sometimes even the primary antibodies have a limited penetration to the synaptic cleft  explain how antibody crosslinking may induce an effect on AMPAR mobility, (2) explain how a drug that blocks desensitization may seem to rescue the enhancement in paired-pulse depression, and (3) contrast the conceptual ideas  that are discussed in Figure 1 of Figure 4A) and what we believe would happen to the AMPARs after crosslinking . We present a synapse with semi-realistic dimensions and appropriate AMPAR spacing. The AMPARs are randomly distributed throughout the synapse and some are clustered in nanodomains as shown in Figure 1. The activation zone of AMPARs is 250 nm in size . One scenario is expected to produce either little (a of 2nd release) or more noticeable paired-pulse depression (b of 2nd release) simply because a different number of AMPARs could be activated. in size  and is r in size . Because in size . For simFigure 4B, the crosslinking condition is represented by primary and secondary particles, or equivalently, large quantum dots, present at the perisynaptic area. The preferential positioning of the antibody complex at perisynaptic zones is based on the papers showing that large macromolecules or particles enter the center of the synaptic cleft of excitatory synapses with greatly decreased frequency (5–10%) (In  (5–10%) . The dep (5–10%) . We do na of 2nd release, lower row) or 10 AMPARs . This lower number is the product of the AMPARs that were closer to the perisynaptic area getting redistributed to the crosslinking complex. This scenario predicts a decrease in the number of activated AMPARs to the second glutamate pulse and an increase in paired-pulse depression that is independent on AMPAR desensitization. The major determining factor in this scenario is the remaining AMPARs present in the center of the synaptic cleft after the crosslinking manipulation.Under these conditions, we predict that a simple antibody-induced redistribution will lead to strong alterations in synaptic transmission. One plausible scenario is that under the crosslinking condition, the first glutamate pulse will activate a lower fraction of AMPARs (about 17) and the second pulse about 13 AMPARs  should be proposed to disentangle the effects of antibody-induced redistribution of AMPARs from the mobility of AMPARs in order to understand the true role of mobility in fast excitatory synaptic transmission. If our ideas are correct they should be testable with available technologies. One such way would be to implement the antibody-induced crosslinking approach to immobilize postsynaptic AMPARs and test the effects of the crosslinking on paired-pulse depression as a function of time. We predict that the effects of the antibody crosslinking approach would have a time-dependent effect, with the crosslinking antibody having weak effects on electrophysiological recordings made shortly after the end of the crosslinking approach (∼30 min) and stronger effects after a longer period has elapse (>1–2 h after crosslinking). These measurements should be complemented with superresolution microscopy (dSTORM on fixed neurons) to colocalize the crosslinked AMPAR complex in relation to the postsynaptic density marker. If we are correct, we predict that the localization experiments may show a time-dependent redistribution of the antibody complex away from the PSD-95-positive cluster and an increase in the staining at extrasynaptic membranes. There may even be an increase in the colocalization of these clusters with the endocytic pits .All of this evidence suggests that better tools are needed that can reduce the mobility of AMPARs without interfering with their distribution or biophysical properties. A plausible way would be to generate knock-in mice where the PDZ binding domain of TARPs is introduced into the GluA1 or GluA2 subunits of the AMPARs. This type of approach was developed in First, a potential starting strategy could be the use of the rapamycin-induced oligomer formation system composed of the FKBP/FRB protein domains  where thin vivo is too slow to participate during periods of repetitive presynaptic stimulation.A second strategy would be to use two of the four known light-inducible dimerization systems described in the literature. The LOV-Jα and the Phy/Pif systems offers the greatest advantage because they have very fast on/off kinetics . These tThe experiments mentioned above should provide new exciting ideas about the function of canonical excitatory synapses. In conclusion, the exciting idea that AMPAR mobility plays a role in tuning fast synaptic transmission deserves further studies.JD and PS evaluate all stages in the preparation of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


BRCA1 and BRCA2 mutation carriers have an increased lifetime risk of developing breast and/or ovarian cancer. Hence, they face the difficult decision of choosing a preventive strategy such as risk-reducing surgeries or intensified breast screening. To help these women during their decision process, several patient decision aids (DA) were developed and evaluated in the last 15 years. Until now, there is no conclusive evidence on the effectiveness of these DA. This study aims 1) to provide the first systematic literature review about DA addressing preventive strategy decisions for female BRCA1 and BRCA2 mutation carriers, 2) to analyze the quality of the existing evidence, 3) to evaluate the effects of DA on decision and information related outcomes, on the actual choice for preventive measure and on health outcomes.Female BRCA1 and BRCA2 mutation carriers, 18 to 75 years, knowledge of test result). The quality of the included randomized controlled trials (RCT) was evaluated with the Cochrane Collaboration’s risk of bias tool. The quality of included one-group pretest-posttest design studies was evaluated with the ROBINS-I tool. Outcomes of included studies were extracted and qualitatively summarized.A systematic literature review was conducted using six electronic databases . One RCT was formally included, but data presentation did not allow for further analyses. The risk of bias was high in three RCT and unclear in one RCT. The risk of bias in the one-group pretest-posttest study was serious. The outcome assessment showed that the main advantages of DA are linked to the actual decision process: Female BRCA1 and BRCA2 mutation carriers during their decision process by significantly improving decision related outcomes. More high-quality evidence is needed to evaluate possible effects on information related outcomes, health outcomes and the actual choice for preventive measures.Decision aids can support female The online version of this article (10.1186/s12911-019-0872-2) contains supplementary material, which is available to authorized users. BRCA1 and BRCA2 [BRCA1 mutation carriers by age 80 years are 72% for BC and 44% for OC. The corresponding estimates for BRCA2 are 69 and 17% [Approximately 0.1 to 0.3% of all women carry a mutation in one of the so-called breast cancer genes nd BRCA2 . These wBRCA1 and BRCA2 mutation is strongly recommended for women with a family history or a personal history of BC and/or OC, which is potentially associated with hereditary mutations [Genetic testing and counselling for a utations . A positBRCA1 and BRCA2 mutation carriers to manage the elevated cancer risk include an intensified breast screening program  as well as risk-reducing surgeries [BRCA1 and BRCA2 mutation, another strategy to reduce the elevated cancer risk is chemoprevention  [BRCA1 and BRCA2 mutation carriers might be reduced by a healthy lifestyle  [Current strategies for female urgeries . To dateurgeries , 9. For ibitors) , 11. Furctivity) , 13.Surgical options include risk-reducing bilateral salpingo-oophorectomy (RR-BSO), risk-reducing bilateral mastectomy (RR-BM) and risk-reducing contralateral mastectomy (RR-CM). The latter is an option for mutation carriers after unilateral BC. The decision for a risk-reducing mastectomy (RR-M) is again followed by a series of upcoming questions, such as considering the surgical technique  and having a breast reconstruction or not. The decision for a RR-BSO is followed by the question of whether receiving hormone replacement therapy (HRT) and, when indicated, which type of HRT should be chosen. Before choosing RR-BSO family planning should be completed.In such complex situations, which require weighing up advantages and disadvantages of options, patient decision aids (DA) might be useful to support individual decision making. DA are tools for people seeking advice. The International Patient Decision Aid Standards (IPDAS) Collaboration defines them as “tools designed to help people participate in decision making about health care options. They provide information on the options and help patients clarify and communicate the personal value they associate with different features of the options” . DA are BRCA1 and BRCA2 gene test is followed by a series of questions and difficult decisions, the need for adequate counseling is high. In this situation the use of a DA might be valuable during the decision-making process. A previous systematic literature review has shown that DA concerning different health decisions when compared to usual care, can improve the consumers’ knowledge and reduce their decisional conflict [Since a positive result of a conflict .BRCA1 and BRCA2 mutation carriers were developed. Although some were evaluated in randomized controlled trials (RCT) or qualitative studies, to date, there is no systematic literature review about the effectiveness of DA for female BRCA1 and BRCA2 mutation carriers.In the last 15 years several DA for female BRCA1 and BRCA2 mutation carriers, to analyze the quality of the existing evidence and to evaluate the effects of DA on decision related outcomes, information related outcomes, actual choice for preventive measure and health outcomes.The aim of this review is to provide the first systematic literature review about the effectiveness of DA for female BRCA1/2. The search strategy was tailored to the requirements of each individual database . They were rejected if the reviewers determined from the title or abstract that the study did not meet the inclusion criteria. After this screening process, full texts were retrieved and further assessed for eligibility independently by two reviewers. Any disagreement was solved by discussion among the reviewers.The quality of RCT was evaluated using the Cochrane Collaborations’ risk of bias tool . This toThe quality of the one-group pretest-posttest design study was evaluated with the Risk Of Bias In Non-randomised Studies - of Interventions (ROBINS-I) assessment tool , which iOne reviewer (LK) extracted the study characteristics and outcome data from the included studies Tables , 3, 4. TAs shown in Fig. BRCA1 and BRCA2 mutation carriers at a point of time, when the women did not know their BRCA1 or BRCA2 test result yet. Despite contacting the authors and receiving more data, we still could not include the study, since the data set provided by the authors did not allow for tracing back the separate study arm in the form needed for our review.The main characteristics of the six included studies are shown in Table The quality analysis of the included RCT –25 showeThe one-group pretest-posttest design study  was rateThe qualitative synthesis of the effectiveness was performed for all included six studies. A variety of outcomes were used to evaluate the effects of the DA. A summary of the outcomes, respective instruments as well as the corresponding main effects of the DA is provided in Tables BRCA1 and BRCA2 mutation carriers can have significant beneficial effects on decision related outcomes. Four studies evaluated the decisional conflict [The qualitative synthesis of the evaluation studies showed that DA for conflict , 25, 27:conflict  and one conflict . One stuFour studies measured the strength of treatment preference –25, 27. Two studies evaluated the outcome “decision satisfaction” , 23. Of Three studies evaluated the influence of the DA on risk perceptions of the affected women , 25, 27,Two studies measured the actual preventive choice made by the included women , 24. OneA variety of instruments were used in the trials to determine health outcomes. Three studies measured distress , 25, 27 One study  measuredAnother study using the IES showed that women in the DA group experienced significantly lower cancer related distress at 6- and 12-month post-randomization compared to the control group . One stuBRCA1 or BRCA2 mutation who are facing the difficult and complex decision of choosing a preventive strategy that goes well with their individual life situation and personal values. Our review gives an overview of the quality of the existing evidence and summarizes the effects of DA on decision, information and health related outcomes as well as on actual choice for preventive measures.This is the first systematic review of the effectiveness of DA for women with a pathogenic Concerning the study quality three of the four RCT showed a high risk of bias, one showed an unclear risk. The strengths of the included RCT were their adequate sequence generation, their complete outcome reporting and the absence of other sources of bias. The main weakness of the identified studies was the insufficient blinding of patients and personnel. Blinding remains challenging in the field of decision support as recipients usually can recognize the assigned intervention. However, options such as providing a DA versus a non-structured set of information materials or flyers should be considered in future studies. Apart from the four RCT, one study with a one-group pretest-posttest design was included, because it fulfilled the inclusion criteria. This study showed a serious risk of bias. Its main deficits were found in the domains confounding and measurement of outcomes. The weak quality of this study is not surprising, since the one-group pretest-posttest design is often criticized because of different threats to its internal and external validity , 29.In summary, the quality of the studies included in this review is compromised to some extent. The results regarding the effects of the DA must be seen in the light of this weakness.BRCA1 and BRCA2 mutation carriers has some advantages with respect to decision related outcomes. Three studies show that the use of a DA leads to significantly less conflict during the decision-making process [We found indications that the use of a DA for female  process , 23, 27. process , 23.Our findings are partly congruent with the results of the most recent Cochrane Collaboration review about the effectiveness of DA for people facing health treatment or screening decisions. In this review 105 RCT involving 31,043 participants were analyzed . The CocConsistent with our results, the Cochrane Collaboration review showed that the use of DA leads to a lower decisional conflict. In contrast to our results, the Cochrane Collaboration review found that the effects on satisfaction are limited. This limited effect is, according to the Cochrane Collaboration review, possibly a consequence of measurement insensitivity: Because satisfaction with usual care is already high, it is difficult to measure differences in satisfaction between DA and control groups. The studies included in the Cochrane Collaboration review used a variety of instruments to measure satisfaction, most of them were not validated. Also, in our review only one of two studies evaluating the effect of the DA on satisfaction used a validated instrument, more precisely the Satisfaction with Decision Scale. Therefore, to make a clear statement about the effects of a DA on satisfaction, more high-quality evidence is needed, including studies using validated and sensitive instruments.BRCA1 and BRCA2 mutation carriers. As younger and middle-aged females have higher levels of health literacy [The main conclusion of the Cochrane Collaboration review is that the largest benefits of DA compared to usual care, are better knowledge and risk perceptions . These rliteracy , 31 and Metcalfe and colleagues  reason tRegarding the actual preventive choice, we found only marginal effects of the use of a DA. Although not reaching statistical significance, Schwartz and colleagues  demonstrBRCA1 and BRCA2 carriers about the “right time” of considering risk-reducing surgery. According to the study, the interviewed women felt that it is necessary to take enough time to deliberate about risk-reducing surgery decisions. Otherwise, they would not feel “ready” and “able” to make these decisions.This argument is supported by a study of Howard and colleagues , who perBRCA1 and BRCA2 mutation carriers who consider various preventive options, is difficult, especially because their decision for or against risk-reducing surgery is very complex and influenced by many factors such as lifetime risk of developing cancer, family history of cancer, having children and age [In contrast to our results, the latest Cochrane Collaboration review on DA showed, excluding Schwartz et al. , a signi and age , 36.BRCA1 and BRCA2 mutation carriers.Therefore, more high-quality evidence is needed to make a clear statement about the effects on the actual preventive choice of preventive measures by the use of a DA for As for health outcomes such as anxiety or distress, two studies revealed an impact of the DA versus the control group which varied over time , 25. OneIn contrast to our results, the effects of DA on health outcomes, such as general health outcomes, anxiety, depression, regret and confidence, in the latest Cochrane Collaboration review were limited . In the Our findings must be considered in the light of the strengths and limitations of this review. The main limitation is that the identified studies showed a high or unclear risk of bias. This might restrict the information value of the validity of DA effectiveness as summarized in this study.Another limitation is that this review only provides a qualitative summary of the outcome results of the included RCT. A meta-analysis was not possible because the identified studies were heterogeneous in terms of study design, time frame between genetic test result disclosure and delivery of the decision aid, follow-up periods and the choice of outcomes. Moreover, we could not access all the required data from all studies –24, despBRCA1 and BRCA2 mutation carriers. Moreover, we sent author request to obtain data in other formats so that they might be included in our review.The strengths of this review are its clear search protocol, the inclusion of six different databases and the involvement of three different reviewers. The critical quality assessment based on the well-established Cochrane Collaborations’ risk of bias tool and the ROBINS-I tool ensures a transparent judgement of studies. We included all types of studies and outcomes in our review, which allows to provide a complete overview of the effectiveness of DA for BRCA1 or BRCA2 mutation. Our work indicates that DA may support female BRCA1 and BRCA2 mutation carriers during their decision process for choosing a preventive measure. This is mainly achieved by improving decision related outcomes. More high-quality evidence is needed to evaluate possible advantages or disadvantages on information related and health outcomes as well as on the actual choice for preventive measures. To provide high-quality evidence and to reach a higher comparability of study results it is important that future research focuses on 1) low-biased study designs and 2) the use of well-established and validated instruments to assess outcomes.This is the first systematic review of the effectiveness of DA for women with a pathogenic Additional file 1:Full search strategy for each database. (DOCX 17 kb)Additional file 2:Quality of randomized controlled trials / Rationale. (XLSX 15 kb)Additional file 3:Quality of pretest-posttest study / Rationale. (XLSX 14 kb)