In awake mice, blocking mGluR1 decreases Cai rises associated with locomotion. In vitro studies and freeze-fracture electron microscopy show that the iGluR-mGluR1 interaction is synergistic and favored by close association of the two classes of receptors. Altogether our results suggest that mGluR1s, acting in synergy with iGluRs, potently contribute to processing cerebellar neuronal signaling under physiological conditions.Type 1 metabotropic glutamate receptors (mGluR1s) are key elements in neuronal signaling. While their function is well documented in slices, requirements for their activation in vivo are poorly understood. We examine this question in adult mice in vivo using 2-photon imaging of cerebellar molecular layer interneurons (MLIs) expressing GCaMP. In anesthetized mice, parallel fiber activation evokes beam-like Ca Although both MLIs and Purkinje cells (PCs) are PV positive, the combined use of specific viral serotype (either AAV2/1 or AAV2/9) and of the human synapsin promoter to drive expression, resulted in the almost exclusive GECI expression in somata and neurites of MLIs  GCaMP3 or GCaMP5 by stereotaxic injections of floxed AAV viral vectors into the cerebellar cortex of adult parvalbumin (PV) Cre mice . An example for a representative MLI soma is displayed in To activate a narrow bundle of PFs, a theta glass pipette was introduced through the meninges until it reached the outermost molecular layer . In ordeentation . Increasi signals elicited by trains of PF stimulation (200 µs-long pulses at 100 Hz) before and after addition of a specific mGluR1 antagonist, CPCCOEt, to the pool bathing the craniotomy. We found that CPCCOEt significantly decreased peak amplitudes for the somatic signals in vivo . The smaller contribution of mGluR1s to the neuropil compared to the somatic signals may reflect the fact that when analyzing the neuropil we have no unambiguous way to discriminate dendrites versus axon. Therefore, our neuropil analysis reflects a mixed population of neurites, with dendrites having a presumably more robust mGluR1 signaling than axonal processes. Nonetheless, analysis of the neuropil allows us to confirm that mGluRs are engaged in dendrites, where the density of parallel fibers synapses is 3 fold higher than in the soma  and quantified by adding the fluorophore Alexa 594 to the drug containing solution . We then compared the mean level of locomotion fluorescence before drug application to the change in locomotion fluorescence after drug application  meaning that a more probable decrease in Cai signal happened after CPCCOEt application if a higher fluorescence response was elicited during locomotion before drug application. These results suggest that under control conditions large signals are more likely to involve mGluR1s than small signals, in accord with the finding that longer or higher frequency PF stimulations are more successful in recruiting mGluR1  occur at the PF-MLI synapse  in control saline and 5 ± 3% (n = 10) in the presence of CPCCOEt, showing that they were mediated by mGluR1. We found that these Cai rises were strongly decreased by addition of CdCl2 to the extracellular solution  at PF-MLI synapses . Given ti signals increases sharply as a function of the duration of presynaptic action potential trains. Secondly, we show that activation of mGluR1s and iGluRs act in synergy to produce Cai rises. Third, we show that the spatial distribution of these receptors at the PF-MLI synapse, as revealed by quantitative EM, favors iGluR/mGluR1 synergy following homosynaptic receptor activation.Three main findings arise from the present work. Firstly, we show that mGluR1s are activated in vivo by synaptically-released glutamate both during electrical stimulation of PFs in anesthetized mice and during enforced locomotion in awake mice. Furthermore, we show that the relative contribution of mGluR1 activation to the postsynaptic Ca2+ permeability . This shows that voltage-gated Ca2+ entry is necessary for mGluR1-activated Cai rises. In this context, our in vivo results showing that mGluR1-dependent rises are totally dependent on simultaneous iGluR activation strongly suggest that AMPAR activation provides a depolarization that activates VGCCs, thus initiating the Cai rise and bootstrapping the mGluR1-activated response. Although most of our experiments used both AMPAR and NMDAR blockers, the evidence shown in 2+-dependence of phospholipase C, which has been documented in cerebellar PCs  in agreement with the European Directive 86/609/EEC and by the ethical committee of the University Paris Descartes.Estimation of sample size was according to published in vivo and in vitro work. Sample size for each experiment is indicated in the main text.Pvalb-Cre mice, were deeply anesthetized with intraperitoneal injection of ketamine (14.8 μg/g)/xylane (10 μg/g) and mounted in a stereotaxic frame. A midline sagittal incision exposed the cranium over the cerebellar vermis. At the site of injection  a 0.5 mm burr hole was drilled and a 34-gauge stainless steel beveled needle (WPI-nanofil) was slowly descended 0.4 mm through a slit cut in the meninges. After a 2 min pause, the needle was retracted 50 μm and then 1.2 μl of AAV2/1.hSyn.Flex.GCaMP3.WPRE.SV40, AAV2/9.hSyn.Flex.GCaMP3.WPRE.SV40  delivering 200 μs long pulses at 30–80 V amplitude. Initial search for stimulation site and parameters was performed with the criteria that 10 pulses at 100 Hz elicited Cafunction .2, 1.5 CaCl2, 10 Hepes, adjusted to a pH of 7.3 with 1 M NaOH solution).Glutamate receptor antagonists GYKI 53655, 5,7-Dichlorokynurenic acid (DCK), CPCCOEt and IEM 1460 were purchased from Tocris or Accent Scientific. Drugs were added to the artificial cerebrospinal fluid solution (ACSF) on the pool bathing the cranial window, which had the following composition (in mM): 150 NaCl, 2.5 KCl, 1 MgCl+ saline designed for slicing of adult mice brain . Slices were maintained for 30–40 min at 34°C in standard recording saline  prior to transfer to the recording set-up. 2PLS imaging was performed at 34°C using an excitation wavelength of 910 nm. Even though precautions were taken to optimize slice survival, a variable fraction of MLIs displayed nuclear fluorescence after slicing. At equivalent expression times, nuclear fluorescence was not observed in vivo, suggesting that changes in the subcellular distribution of the GCaMP protein are due to the slicing procedure. Such changes are indicative of deleterious effects of slicing and therefore emphasize the importance of in vivo studies. MLIs were studied only if their nucleus did not show any fluorescence. Imaging was performed at depths of 40 to 70 μm from the slice surface. Imaging and extracellular stimulation were as described in One to three weeks after stereotaxic injection, sagittal cerebellar slices were prepared in sucrose-containing saline as described previously  or usingce brain  with the3, 25 glucose, 2 CaCl2, 0.1 MgCl2 and 10 HEPES, pH 7.4 with NaOH. WCR was performed in MLIs with an intracellular solution containing (in mM): 140 Cs Gluconate, 4 MgCl2, 10 HEPES-K, 0.4 Na-GTP, 4 Na-ATP and 100 μM spermine, pH 7.3 with CsOH. MNI-glutamate (0.9 mM) was added to the recording chamber and photolysis was achieved with 100 μs pulses from a 405 nm laser as described elsewhere  issued from a cross of mice carrying the GCaMP6f transgene in the Igs7 locus (Jackson labs stock number 024107) and Pvalb-Cre mice. Expression pattern was similar to that obtained with viral injections.For whole-cell recording (WCR) experiments, slices from mice aged 30–35 days were prepared as described above. Extracellular recording saline contained (in mM): 135 NaCl, 4 KCl, 2 NaHCOlsewhere . CurrentIn vivo experiments were usually performed 2–3 days in a row in a week depending on the availability of the transgenic mice and the success of viral injection. In each animal, all activated neurons within the focal plane were sampled. Biological replicates were the number of animals used in the experiments. Technical replicates were the number of repeated stimulations for each condition. Outliers were not excluded from the data pool.2+ . Background fluorescence Fb(t) was estimated as the average fluorescence of 20–30 pixels with the lowest F value throughout all the frames of the sequence. A somatic ROI was defined by a polygon drawn along the contour of the ring-shaped somatic pre-stimulus fluorescence. F(t) was calculated as the time course of the averaged fluorescence from all pixels in a ROI. ΔF/Fo was computed as (F(t)-Fo)/(Fo-FB). Fo and FB are the averages of F(t) and Fb(t) over the pre-stimulus period respectively. Values for pooled data are given as mean ± s.e.m. When assessing drug effects, results from GCaMP3 and GCaMP5 were pooled together, because these two GECIs have similar affinities for Ca2+ . For staThree weeks after stereotaxic injection, mice were prepared for imaging during locomotion by implanting a chronic window and fixing a head post. Before surgery (10–15 min), mice were injected with buprenorphine  for pain reduction. Lidocaine was used as a local anesthetic. Surgeries were performed under isoflurane (1.5–2.0% by volume in air) to maintain a surgical plane of anesthesia as determined by non-responsiveness to toe pinch. Eye ointment was applied under anesthesia at the start of surgery. Animals were held in a stereotaxic apparatus using ear and nose bars to stabilize the head. Body temperature was set with a heating pad and controller using biofeedback from a rectal thermocouple.For the surgical procedure, the area of the head over the cerebellum was first shaved and the underlying skin was treated repeatedly with ethanol (70%) and Betadine in alternation. The skin above the skull was removed and the opening of the periosteum as well as any membranous tissue were cleaned. Once the skull was dry, a cranial window was carefully drilled using a handheld high-speed micromotor drill (Foredom). The center of the craniotomoy was located ~2 mm from lambda on the midline targeting lobule IV/V of the cerebellar vermis. Skull removal was performed under application of an ACSF solution to minimize the risk of damaging the dura overlying the brain. After skull removal, the ACSF solution was used to clean any debris from the brain surface and to maintain the brain moist providing an interface to the chronic window. A chronic window was fabricated from a cover glass  laser cut to a small oval shape (2.5 mm x 2 mm) to better fit the cranial opening, and to feature two holes of ~500 μm diameter and 1.5 mm separation on the major axis for drug delivery. The window was placed over the cerebellar vermis lobule IV/V and kept in direct contact with the brain by lowering the window with the help of a needle mounted in the stereotaxic setup. The chronic window was fixed in place with dental cement (Super-Bond C and B). After setting of the dental cement, the bone surrounding the craniotomy was thinned with the micromotor drill to allow for better access with the 20x objective. Then a custom-made stainless steel head post with a central opening for the craniotomy was cemented in place with dental cement (Super-Bond C and B). The chronic window was covered with silicone for protective purposes after surgery. Mice were carefully monitored during surgical recovery, with all animals showing normal behavior including the absence of motor deficits. Mice were allowed to recover at least 2 days before proceeding to the locomotion and imaging experiments with ad libitum access to food and water.A sketch of the imaging setup can be seen in 2) the wheel at 7 s until a maximal speed of 10 cm/s was reached at 10 s. This rotation speed was kept constant until 23 s at which point the motor linearly decelerated (−3.33 cm/s2) until standstill at 26 s and then moved out of the gear on the treadmill axis allowing for self-paced walking. The rotation of the treadmill was recorded with a rotary encoder  at 40 kHz with a standard DAQ acquisition board . Mice were monitored with infra-red (940 nm LED illumination) videography at 200 frames/s  during the entire recording sessions.Imaging recordings lasted for 40 s and were repeated every 2–4 min. The speed and the duration of locomotion during the recordings was imposed in an attempt to minimize variations across recordings. The forced locomotion scheme was as follows: Recordings started with a 6 s long period of baseline during which the mouse was able to freely rotate the treadmill. At 6 s, a servo motor moved a continuous rotation servo in a gear attached to the axis of the treadmill. The continuous servo started to linearly accelerates  was exchanged with a ACSF containing additionally 100  or 200 μM CPCCOEt . Two holes of ~500 μm diameter were drilled in the cover-slips prior to implantation to allow for drug access. To further improve drug access, the dura mater was pierced with a sharp glass pipette before the experiment under anesthesia (1.5% isoflurane in air). In a subset of experiments (4 out of 5 recordings), 50 μM Alexa 594 was added to the drug-containing ACSF to assess the dynamics of drug diffusion in the recorded brain area . 50 μM AFluorescence recordings during forced-locomotion before- and after drug application were performed in 4 mice in total. One of the animals was recorded on two different days . The first recording of animal #1, animal#1A, was removed from all further analysis based on the lack of fast Alexa fluorescence increase . The fouAoverlap/Aunion with Aoverlap = Abefore drug ∩ Aafter drug and Aunion = Abefore drug∪ Aafter drug. The fluorescent traces of overlapping ROIs were then compared between the before and after drug condition.The pre-processing of all raw calcium imaging data – image registration and ROI detection – was done using Suite2p using default settings . ROI idei,jF = i,jFMLI- 0.7 i,jFNeuropil . Subsequently, fluorescence traces were normalized using the pre-motorization baseline period (< 5 s). Blocks of recordings were scanned for a recording j = k with the least active baseline period based on the rotary encoder recordings, that is, the recording j = k for which the animal showed the least amount of locomotor activity during the first 5 s. Fluorescence during that least active baseline, referred to as restF, was averaged for each ROI following i denotes the index of the respective ROI and brackets <> indicate the temporal averaging. ROI fluorescence traces of a block of recordings j were then normalized using this baseline activity according to Cellular fluorescence traces were first compensated for surrounding neuropil signal by subtracting a scaled-down version, that is, th percentile of j denotes pre-drug recording numbers as linearly increasing integers; A, B, C are fit parameters) to the pre-drug locomotion fluorescence values. This function is subtracted from all recordings, pre- and after-drug, which normalized the pre-drug locomotion fluorescence to zero and revealed any drug induced change beyond the general decay .All analysis scripts are available online  and transcardially perfused by cold Phosphate Buffer Saline (PBS) followed by PBS containing 4% of paraformaldehyde (PFA). After decapitation the cerebellum was removed and post fixed in PBS PFA solution for 2 hr. 50 μm thick parasagittal slices were prepared from the vermis using a vibrating slicer . The slices were first incubated in PBS containing 0.3% Triton and 10% fetal bovine serum for 5 hr at room temperature then at 4°C overnight with a mixture of rabbit polyclonal serum anti-Parvalbumin  and guinea pig polyclonal serum anti calbindin D28k  diluted to 1/1000 in PBS containing 1 mg/ml of Bovine Serum Albumine (BSA). After 3 washes in PBS, the slices were incubated for 3 hr with secondary goat antibodies anti-rabbit IgG and anti-guinea pig IgG conjugated to Alexa fluor 546 and Alexa fluor 647 respectively . Slices were mounted between slides and cover slip with Vectashield Mounting Medium . Stack of images (0.44 µm thickness) were acquired using an LSM 510 confocal microscope  equipped with Plan-Neofluar 40x/1.3 oil objective and appropriate excitation  and emission filters . Image analysis and projections were done by LSM and ImageJ Software.SDS-FRL was performed as described previously . Adult W In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Bao et al. examined whether type 1 metabotropic glutamate receptors (mGluR1s) are engaged at the parallel fiber-molecular layer interneuron synapses in the cerebellum during physiological activity in awake behaving mice. Through a combination of in vivo calcium imaging, slice electrophysiology and freeze-fracture electron microscopy, the authors demonstrate the recruitment of mGluR1-dependent calcium signals during locomotion, and that mGluR1 activation is synergistic with the activation of ionotropic glutamate receptors to produce the rise in intracellular calcium.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by a Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Marco Capogna (Reviewer #2).Thank you for submitting your work entitled "Synergistic action of metabotropic and ionotropic glutamate receptors in cerebellar molecular layer interneurons in vivo" for consideration by eLife's policy to reject manuscripts that are likely to require more than 2 months of additional work, the decision was made to reject this version so that you are free to submit it elsewhere at this time, should you wish. Nonetheless, all the reviewers and editors see the very high potential value and interest of the work, and if you agree that doing the requested experiments will strengthen the manuscript, eLife would welcome a new submission of this work. The authors are encouraged to take the time to address the concerns thoroughly.All three reviewers agree that the study is of high potential interest. However, major issues have been raised such that the conclusions are not firmly supported by the data shown. Because it is Reviewer #1:The study addresses physiological requirements for activation of group 1 mGluRs and their functional and spatial relationship to AMPARs at the parallel fiber-molecular layer interneuron (MLI) synapses in the cerebellum in vivo. Using GCaMP Ca imaging of MLI neurons, the authors report that longer duration PF stimulation (1 s rather than 200 ms) at 100 Hz is more effective in eliciting mGluR1-dependent Ca rises in MLI soma, in a manner that is synergistic with activation of AMPARs. Interestingly, AMPARs involved are not Ca-permeable in contrast to previous studies at developing MLI synapses, and the depolarization caused by AMPAR activation likely recruits VGCC to confer VGCC-dependence of mGluR1 activation as shown in acute slices. The authors also perform ultrastructural analysis of mGluR1 subcellular distribution by freeze-fracture replica labelling, which shows its localization close to the PSD. Altogether, in identifying several features of mGluR1 involvement at PF-MLI synapses in vivo, while somewhat descriptive, the findings are valuable. The major technical issue is the use of different GCaMP variants. While the initial demonstration of sensory stimuli-evoked MLI activity and its similarity to PF stimulation-triggered response are shown by GCaMP6s, subsequent analyses use mostly GCaMP3 imaging which not only differ in sensitivity for detecting MLI activity but also display different kinetics. In addition, demonstration of the involvement of mGluR1 for 100 ms-long trains at 500 Hz is shown using GCaMP5. There needs to be a consistency in the activity readout used across experiments to compellingly demonstrate that mGluR1 activity as reported is involved in vivo under physiologically relevant paradigms.Reviewer #2:This work directed by Isabel Llano and Ryuchi Shigemoto investigates parallel fibers (PFs) glutamatergic synapses onto molecular layer interneurons (MLI) of the mouse cerebellum. They found that mGluR1s are activated in vivo by synaptically-released glutamate and act together with ionotropic (i)GluRs to trigger intracellular calcium rise. Furthermore, and this is one of the nicest findings of this manuscript, high resolution analysis of the spatial distribution of postsynaptic mGluR1 PF-MLI synapse distribution show that these receptors are remarkable close to iGluRs, consistent with their observed synergic functional effects.The manuscript is competently written and illustrated. The senior authors are expert researchers in the field of cerebellum synapses, calcium imaging and high resolution receptor localization analysis.Overall, I like this manuscript. I have some constructive criticism that I hope will help the Authors to improve the quality and completeness of their study and ultimately enhance the impact of their manuscript when it will be published.1) It is a pity that most of the study uses electrical stimulation of PFs, and not a more physiological way to stimulate the PFs-MLI pathway, to investigate the calcium signaling in MLIs and iGluRs/mGluR1 synergic activation. In a few experiments, calcium responses of MLI are evoked by air puffs. However, due to the relatively high calcium signal variability observed after air puff stimulation, the Authors decided to conduct most of their pharmacological experiments by using electrical stimulation. On the contrary, I recommend the Authors to reproduce key results  by using air puffs stimulation, and to include the results of these new experiments in the revised version of the manuscript. Moreover, it would be extremely interesting to investigate the PFs-MLI synapses and the iGluRs/mGluR1 signaling evoked by eyeblink conditioning, a form of classical conditioned learning, that overwhelming evidence demonstrate to involve cerebellar synapses as major substrate. In my opinion to include the requested new experiments (more air puff data and eyeblink conditioning) would enhance very significantly the significance and the impact of this work.2) Drugs are applied to the pool bathing the craniotomy and no wash out is possible and observed. Furthermore, the actual concentration of the drugs reaching the receptors remains unknown. It would useful to perform some control experiment  to be reassured that the lack of the action of some drugs  is a genuine result and not (at least in part) due to poor drug diffusion to plasma membranes containing AMPARs.Reviewer #3:2+ transients from MLIs in vivo, and also in ex vivo slices. These are important questions and innovative techniques are employed to address them, but the data are preliminary which leaves the conclusions poorly supported.The manuscript by Bao et al. seeks to find out whether mGluRs are engaged on Molecular Layer Interneurons (MLIs) of the Cerebellum during physiologically relevant synaptic activation in vivo and whether their signalling is dependent on iGluR activity. To answer these questions, the authors perform novel and technically demanding recordings of CaMajor comments:1) It is claimed that MLIs are activated as a "beam" by air puffs. This is not quantified so conclusions on this question are premature. This also questions whether it is reasonable to replicate the physiological stimulation with electrical stimulation of the PFs.2) The observed timecourse of the putative mGluR1-mediated calcium response is very rapid for a G-protein induced response . Furthermore, the timecourse seems to be exactly the same for both mGluR1-mediated and iGluR-mediated components. This doesn't seem likely which calls into question whether the responses are indeed mediated by mGluR1. To confirm this requires more than just a single drug application, which could in fact be inhibiting glutamate release from PFs.3) The rationale for the use of direct PF stimulation is that it gives a more reliable signal than air puffs and that they are equivalent. However, the signals are not that similar despite the assertion. Decay rates much slower for air puffs. Furthermore the most equivalent stimulation is 20 pulses at 100Hz but at this stimulation duration and frequency there is little effect of mGluR1.4) One of the core findings claimed in the manuscript is the synergy between iGluR and mGluR but this is never directly demonstrated. Need to compare application of iGluR antagonist and then mGluR antagonist vs mGluR antagonist alone and quantify the difference.5) Overall there is reasonable evidence for lack of Ca-permeable AMPARs but it would be useful to show a positive control for IEM use in vivo. What does it do in vitro?6) The experiment to test role of VGCCs is very preliminary. Photorelease of Glu onto the soma likely initiates APs leading to the observed calcium signal. The fact this is blocked in a single experiment is not convincing and in any case does not reveal much about whether the AMPARs are calcium permeable. The claim that "response of MLIs to mGluR1 agonists is abolished by inclusion of VGCC blocker Cd2+" is not supported by the data.2+-dependent mechanisms.7) An alternative possibility for synergy between iGluR and mGluR is electrotonic effects on glutamate diffusion . However, insufficient evidence is presented to distinguish between this and CaeLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Gary Westbrook as the Senior Editor The following individual involved in review of your submission has agreed to reveal their identity: Marco Capogna (Reviewer #2). The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Thank you for resubmitting your article "Synergism of type1 metabotropic and ionotropic glutamate receptors in cerebellar molecular layer interneurons in vivo" for consideration by eLife papers without additional data, even if they feel that additional data would make the manuscript stronger.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as Summary:This is a revision of a previously submitted study, examining the activation of group 1 mGluRs at parallel fiber-molecular layer interneuron (MLI) synapses in the cerebellum during physiological activity, now monitored in awake behaving mice. The authors demonstrate recruitment of mGluR-dependent Ca signals during locomotion, and that mGluR activation is synergistic with the activation of iGluRs.Although the two reviewers of the original submission found that the revision suitably addressed their points, the third reviewer who was new, had some concerns, especially pointing out that although demonstrating the activation of mGluRs during walking was novel, whether this resulted from PF activity similar to the electrical stimulation used in anesthetized mice and in other slice physiology experiments remained unclear. Upon discussion amongst all three reviewers and the senior editor, we request that the authors carefully address the following points by making use of data or information that they might already possess, by toning down the claims or by referencing existing literature.1) Information about the intensity of the electrical stimulation used in anesthetized mice experiments and how it relates to the data acquired in awake behaving mice would be valuable. That is, how many PFs are being recruited? What EPSC amplitudes are associated with the reported calcium transients?2) Reword the text inciting anticipation that the study addresses a specific role for mGluRs in synaptic plasticity, for example at the start of the Abstract.3) The freeze-fracture EM data is very nice and convincing. However, many of the measurements in the manuscript are taken from the cell body where surely few excitatory synapses are found. The authors should acknowledge this issue in the text.4) It would be nice to have more details about the location of the imaging, i.e. how deep into the molecular layer. Having this information would help the reader determine the type of cells imaged because there could be functional heterogeneity between cells localized in different parts of the molecular layer.5) Figure 2D and 2E. If the bar at the very left in both panels represent the same dataset, this should be stated in the legend. [Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]Reviewer #1:[…] The authors also perform ultrastructural analysis of mGluR1 subcellular distribution by freeze-fracture replica labelling, which shows its localization close to the PSD. Altogether, in identifying several features of mGluR1 involvement at PF-MLI synapses in vivo, while somewhat descriptive, the findings are valuable. The major technical issue is the use of different GCaMP variants. While the initial demonstration of sensory stimuli-evoked MLI activity and its similarity to PF stimulation-triggered response are shown by GCaMP6s, subsequent analyses use mostly GCaMP3 imaging which not only differ in sensitivity for detecting MLI activity but also display different kinetics. In addition, demonstration of the involvement of mGluR1 for 100 ms-long trains at 500 Hz is shown using GCaMP5. There needs to be a consistency in the activity readout used across experiments to compellingly demonstrate that mGluR1 activity as reported is involved in vivo under physiologically relevant paradigms.We thank this reviewer for her/his appreciating the value of our work. The main objection raised by this review was that “While the initial demonstration of sensory stimuli-evoked MLI activity and its similarity to PF stimulation-triggered response are shown by GCaMP6s, subsequent analyses use mostly GCaMP3 imaging which not only differ in sensitivity for detecting MLI activity but also display different kinetics.” To address this criticism, we have removed data using GCaMP6 from this ms. The new set of experiments in behaving mice  were performed in animals expressing GCaMP3, that is, the same protein as that used for the initial characterization of the effect of mGluR block on PF-evoked Cai rises. These new experiments demonstrate that mGluRs are activated in awake and physiologically relevant conditions.Reviewer #2:[…] Overall, I like this manuscript. I have some constructive criticism that I hope will help the Authors to improve the quality and completeness of their study and ultimately enhance the impact of their manuscript when it will be published.1) It is a pity that most of the study uses electrical stimulation of PFs, and not a more physiological way to stimulate the PFs-MLI pathway, to investigate the calcium signaling in MLIs and iGluRs/mGluR1 synergic activation. In a few experiments, calcium responses of MLI are evoked by air puffs. However, due to the relatively high calcium signal variability observed after air puff stimulation, the Authors decided to conduct most of their pharmacological experiments by using electrical stimulation. On the contrary, I recommend the Authors to reproduce key results  by using air puffs stimulation, and to include the results of these new experiments in the revised version of the manuscript. Moreover, it would be extremely interesting to investigate the PFs-MLI synapses and the iGluRs/mGluR1 signaling evoked by eyeblink conditioning, a form of classical conditioned learning, that overwhelming evidence demonstrate to involve cerebellar synapses as major substrate. In my opinion to include the requested new experiments (more air puff data and eyeblink conditioning) would enhance very significantly the significance and the impact of this work.To deal with this issue, we searched for an experimental paradigm that allows us to perform 2-photon calcium imaging in behaving mice. While the suggestion of the reviewer to study signals “evoked by eyeblink conditioning” is interesting, we preferred to continue working in the same cerebellar region as that used during the studies with electrical stimulation of PFs. This choice is dictated by the need to decrease biological variability, already large in a behaving situation. We decided to a conduct pharmacological study on the effect of mGluR1 block on the calcium signals recorded from MLIs while mice walk in a wheel since walking was previously shown to engage MLI activity in the vermis both with electrophysiological recordings  and with calcium imaging . The results of these experiments are described in section “Activation of mGluRs in MLIs during locomotion” .The data show that mGluR1 block decreases the magnitude of the calcium signals associated with walking. Furthermore, the amount of inhibition depends on the initial amplitude of the signal, which is in accord with the results obtained with electrical stimulation of PFs.2) Drugs are applied to the pool bathing the craniotomy and no wash out is possible and observed. Furthermore, the actual concentration of the drugs reaching the receptors remains unknown. It would useful to perform some control experiment  to be reassured that the lack of the action of some drugs  is a genuine result and not (at least in part) due to poor drug diffusion to plasma membranes containing AMPARs.It is true that in vivo recordings do not offer the same level of quantification as does slice work. The situation is not well suited to perform dose-response curves. However, we do have several lines of evidence indicating that the drugs reach the molecular layer at sufficiently high concentrations to achieve the desired block. Concerning experiments on anesthetized mice, as shown in Figure 2, addition of ionotropic blocker after mGluR block totally abolishes the calcium signals elected by PF stimulation.Concerning the new experiments in behaving mice, we added Alexa 594 to the craniotomy and followed the time course of fluorescence increase. The results are shown in Figure 3—figure supplement 1 and indicate that the Alexa concentration equilibrates with a time constant of 5 minutes. Since Alexa and CPCCOEt have similar molecular weights, the time constant of equilibration of CPCCOEt is likely close to 5 minutes as well.Reviewer #3:The manuscript by Bao et al. seeks to find out whether mGluRs are engaged on Molecular Layer Interneurons (MLIs) of the Cerebellum during physiologically relevant synaptic activation in vivo and whether their signalling is dependent on iGluR activity. To answer these questions, the authors perform novel and technically demanding recordings of Ca2+ transients from MLIs in vivo, and also in ex vivo slices. These are important questions and innovative techniques are employed to address them, but the data are preliminary which leaves the conclusions poorly supported.We thank the reviewer for her/his review of our work. The general objection of the reviewer is that data is preliminary: “, but the data are preliminary which leaves the conclusions poorly supported.” This statement was primarily directed to the air puff experiments, which have been removed. For the rest of the manuscript, we stress that our initial presentation of our results was very succinct, thus giving perhaps a false impression of superficiality. In fact our work, as presented in the original manuscript, strongly supports the conclusion that mGluRs type 1 are engaged in MLIs following PF stimulation in anesthetized mice. The new experiments that have now been incorporated to the revised version show that these receptors are also recruited during walking in awake mice. Furthermore, the manuscript investigates details of iGluR-mGluR interactions, and it presents for the first time the distribution of mGluR1 at the PF-MLI synapse, using quantitative EM. Altogether, the work presents a complete description of the mGluR1 component of PF-MLI synapses, all the way from the molecular level up to awake in vivo data. In the revised version, we have added some quantification and experimental detail to document better the experimental bases of our conclusions.Major comments:1) It is claimed that MLIs are activated as a "beam" by air puffs. This is not quantified so conclusions on this question are premature. This also questions whether it is reasonable to replicate the physiological stimulation with electrical stimulation of the PFs.This data set has been removed.2) The observed timecourse of the putative mGluR1-mediated calcium response is very rapid for a G-protein induced response . Furthermore, the timecourse seems to be exactly the same for both mGluR1-mediated and iGluR-mediated components. This doesn't seem likely which calls into question whether the responses are indeed mediated by mGluR1. To confirm this requires more than just a single drug application, which could in fact be inhibiting glutamate release from PFs.Concerning the speed of the responses: G-protein induced responses vary considerably in their onset kinetics. Our own work on MLIs has shown that mGluR1-induced currents and mGluR1- induced calcium rises in these cells can rise within a few tens of ms after application of a glutamatergic agonist . Such a time course is compatible with the time course of calcium rises observed in the present work.Concerning the comparison of response kinetics with and without blockers: Because we are using a calcium probe with relatively slow kinetics, the time course of the calcium rises is largely dictated by the probe and is unlikely to give us insight into the receptors and/or signaling pathways involved. This is the reason that we have refrained from time course considerations.Concerning the drug application, which could in fact be inhibiting glutamate release from PFs.:We are not aware of any evidence for mGluR1 being expressed at the presynaptic side of central synapses. R Shigemoto’s laboratory has looked at different stages of development and found no immunoreactivity for mGluR1 at PF axons . On the other hand, block of mGluR1 reduces the LTD induced at PF-stellate cell synapses with certain stimulation protocols . As endocannabinoid production in PCs is enhanced by mGluR1 activation, the LTD is thought to involve an inhibition of glutamate release by endocannabinoids receptors present in PFs. Although we have no evidence for LTD induction in our work, one could argue that there is an endogenous level of LTD at this synapse that is modulated by endocannabinoids. However, block of mGluR1 would be expected to decrease endocannabinoid production and then increase glutamate release. Therefore, this mechanism has the wrong sign to explain the observed inhibition associated with mGluR1 block.3) The rationale for the use of direct PF stimulation is that it gives a more reliable signal than air puffs and that they are equivalent. However, the signals are not that similar despite the assertion. Decay rates much slower for air puffs. Furthermore the most equivalent stimulation is 20 pulses at 100Hz but at this stimulation duration and frequency there is little effect of mGluR1.The comparison does not longer apply, as the data set from air puff experiments has been removed.4) One of the core findings claimed in the manuscript is the synergy between iGluR and mGluR but this is never directly demonstrated. Need to compare application of iGluR antagonist and then mGluR antagonist vs mGluR antagonist alone and quantify the difference.We agree that our rationale was not explained correctly and we added an explicit comparison for the percentage of block observed with iGluRs versus mGluR1 antagonists. Concerning the suggestion of blocking mGluR1 in the presence of iGluRs antagonists, as shown in Figure 4B, blocking iGluRs results in an almost total block of the calcium rises evoked by PF stimulation (>90 % block at 100 Hz during 1 s), so that it is impractical to study the pharmacology of the remaining response. By contrast, applying CPCCOEt alone inhibits the response by approximately 50 % , and the remaining response is abolished by iGluR blockers .5) Overall there is reasonable evidence for lack of Ca-permeable AMPARs but it would be useful to show a positive control for IEM use in vivo. What does it do in vitro?We have no positive control for this drug in vivo. However, the fact that calcium rises are abolished after addition of mGluR1 and iGluRs blockers  indicates that drugs do reach the molecular layer. Furthermore, the new data set with ALEXA  gives us a time constant of 5 minutes for diffusion of this fluorophore in the tissue. These results suggest that IEM, as well as the other drugs used, did reach the imaged site. And given the published effect of IEM in vitro , the absence of effect on the calcium rises can be interpreted as indicative of lack of calcium-permeable AMPA Rs.6) The experiment to test role of VGCCs is very preliminary. Photorelease of Glu onto the soma likely initiates APs leading to the observed calcium signal. The fact this is blocked in a single experiment is not convincing and in any case does not reveal much about whether the AMPARs are calcium permeable. The claim that "response of MLIs to mGluR1 agonists is abolished by inclusion of VGCC blocker Cd2+" is not supported by the data.2+-permeable AMPARs”.We conducted new experiments in slices to verify that the signals evoked by 1ms glutamate uncaging over the soma are due to mGluR1 activation. The results were clear, with robust response in control saline and hardy any signal in the presence of the mGluR1 blocker. This is now reported in subsection “Synergy between iGluRs and mGluR1s is not mediated by Ca2+-permeable AMPARs”).Concerning the block by Cd, the block shown in Figure 4C is one example that is representative of 4 experiments as stated in the text (subsection “Synergy between iGluRs and mGluR1s is not mediated by Ca7) An alternative possibility for synergy between iGluR and mGluR is electrotonic effects on glutamate diffusion . However, insufficient evidence is presented to distinguish between this and Ca2+-dependent mechanisms.i rises following combined activation of mGluR1s and iGluRs”).We added a paragraph to discuss this possibility  Information about the intensity of the electrical stimulation used in anesthetized mice experiments and how it relates to the data acquired in awake behaving mice would be valuable. That is, how many PFs are being recruited? What EPSC amplitudes are associated with the reported calcium transients?The intensity of the extracellular stimulation  as well as the criteria for choosing the stimulation voltage are described in Materials and methods as:i rises whose trial-totrial variability did not exceed 10 %.”“The pipette was placed on the superficial molecular layer more than 150 µm from site imaged to avoid direct stimulation of the neurons in that field. Ag-AgCl electrodes connected the pipette to an isolated pulse stimulator (AM-systems) delivering 200 µs long pulses at 30-80 V amplitude. Initial search for stimulation site and parameters was performed with the criteria that 10 pulses at 100 Hz elicited CaTo estimate how comparable are signals recorded with parallel fiber (PF) stimulation protocol and those during locomotion, we first compared the calcium transients evoked in these two types of experiments. For 0.2 to 1 sec long PF trains at 100 Hz, we obtained peak DF/Fo of 114+31 and 347+43 % (mean+sem) respectively, whereas for the locomotion associated transients the corresponding values were 128.2+8.5 %. Thus, calcium transients in MLIs somata are in a comparable range for both experimental conditions. Secondly, as the reviewers suggested, we can estimate the EPSC in MLIs under these two conditions. We have data from Jin Bao’s paper in Journal of Neuroscience (2010) reporting in stellate cells an EPSC amplitude of ~100 pA following stimulation of GCs with theta glass pipettes, as used in the current study. This value is comparable to that obtained from in vivo patch clamp recordings of stellate cells in locomoting mice . However, spatial and temporal patterns of activation differ. The electrical stimulation we use imposes a “beam” like pattern of MLI recruitment . On the other hand, locomotion engages a much larger population of MLIs  in accord with results from Ozden et al., 2012. Temporal differences are also expected, as discussed below.Concerning the number of PFs recruited: In extracellular stimulation experiments, we presume that action potential thresholds are rather homogeneous across PFs, so that all PFs within a beam are excited at the frequency imposed by extracellular stimulation. During locomotion, likewise, a vast majority of GCs are activated . The impact on MLIs is a change in firing rate from an average value of 20 Hz in quiet mice to 60 Hz during self-paced locomotion . GC firing in mice walking spontaneously is very heterogeneous in time and in space. In time, firing occurs in high frequency bursts with frequency within a burst ranging from 60 to 142 Hz, and burst durations ranging from 30 to 140 ms. Strikingly, these values are close to those needed to obtain measurable mGluR responses with extracellular stimulation . In space, individual GCs are reported to vary markedly in their firing patterns when mice are walking. In view of the results of Figure 3 indicating a measurable mGluR contribution in a rather small proportion of MLIs, it is plausible that particularly active GCs cooperate within the dendritic field of these particular MLIs to produce mGluR activation as a result of temporal and spatial summation of the perisynaptic glutamate concentration.We added a slightly shorter version of these arguments to Discussion.2) Reword the text inciting anticipation that the study addresses a specific role for mGluRs in synaptic plasticity, for example at the start of the Abstract.We deleted from the Abstract the mention of synaptic plasticity. We also removed from Introduction the sentence referring to “other forms of synaptic plasticity”.3) The freeze-fracture EM data is very nice and convincing. However, many of the measurements in the manuscript are taken from the cell body where surely few excitatory synapses are found. The authors should acknowledge this issue in the text.We acknowledge now this difference in the context of presenting the results of mGluR block on the neuropil as follows:“Nonetheless, analysis of the neuropil allows us to confirm that mGluRs are engaged in dendrites, where the density of parallel fibers synapses is 3 fold higher than in the soma .”4) It would be nice to have more details about the location of the imaging, i.e. how deep into the molecular layer. Having this information would help the reader determine the type of cells imaged because there could be functional heterogeneity between cells localized in different parts of the molecular layer.Imaging depth ranged from 50 to 100 µm from the surface . In both experimental paradigms we imaged at depths at which the MLI population will comprise stellate cells.5) Figure 2D and 2E. If the bar at the very left in both panels represent the same dataset, this should be stated in the legend.Added to Figure 2E legend: “The 100 Hz, 0.1 s bin is derived from the same data set as that used for the first bin in panel D.”


Altogether, our findings suggest an anxiolytic effect of NREM sleep, and indicate a potential therapeutic strategy for anxiety.Social defeat stress (SDS) plays a major role in the pathogenesis of psychiatric disorders like anxiety and depression. Sleep is generally considered to involve recovery of the brain from prior experience during wakefulness and is altered after acute SDS. However, the effect of acute SDS on sleep/wake behavior in mice varies between studies. In addition, whether sleep changes in response to stress contribute to anxiety is not well established. Here, we first investigated the effects of acute SDS on sleep/wake states in the active period in mice. Our results showed that total sleep time (time in rapid eye-movement [REM] and non-REM [NREM] sleep) increased in the active period after acute SDS. NREM sleep increased mainly during the first 3 h after SDS, while REM sleep increased at a later time. Then, we demonstrated that the increased NREM sleep had an anxiolytic benefit in acute SDS. Mice deprived of sleep for 1 h or 3 h after acute SDS remained in a highly anxious state, while in mice with  Frustration in social interactions is a common problem, most often illustrated by bullying in schools. Social defeat is a major source of stress in humans and plays an important role in the pathogenesis of mental disorders such as anxiety and depression . At the In addition to causing affective disorders, acute stress promotes wakefulness and enhances arousal while the stressor persists . Social Rodent models of SDS are based on a resident-intruder paradigm where the intruder mouse is defeated by an aggressive resident CD-1 mouse, and they have been shown to exhibit behavioral changes –22 and sad libitum food and water. All experiments were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals of Zhejiang University and all protocols were approved by the Institutional Animal Care Committee (ZJU2015-015-01).Male C57BL/6J mice 8–10 weeks old were maintained under standard housing conditions on corn cob litter in an animal room with controlled temperature (22 ± 1°C) and humidity (55% ± 5%) on a 12-h light/dark cycle  with Each mouse was anesthetized with ketamine and xylazine  and placed in a stereotaxic instrument . The mice used for sleep recording were implanted with a custom-made EEG and EMG unit placed on the rear of the skull. EEG signals were recorded from electrodes on the frontal cortices . Two stainless-steel wires were inserted into neck muscles as EMG electrodes. The EEG electrodes and two stainless-steel screws for anchorage were fixed to the skull with dental cement. Tissue glue  was used to help heal wounds and fix the EEG-EMG electrodes. Mice were allowed to recover for 1 week and briefly handled for 5 min daily for habituation.ad libitum sleep or sleep deprivation.Male CD-1 retired breeder mice 6–8 months old were purchased from Charles River Co.  and housed singly with 3 days of screening to select those meeting the social defeat criterion . UnsuitaAfter 1 week of recovery in single housing, mice were transferred to a recording cage in a sound-attenuated box and habituated to the recording cables for 3 days. To allow free movement in the recording cage without tangling, a slip-ring device  was used for the cable. The EEG and EMG signals were amplified and filtered  by an AC amplifier , digitized at 200 Hz , and recorded by LabChart software (AD Instruments).The sleep analysis software SleepSign  was used for sleep state scoring. All scoring was automatic on the basis of the EEG and EMG waveforms in each 4-s epoch. Sleep state was defined as follows: wakefulness, desynchronized EEG and high EMG activity; NREM sleep, synchronized EEG with high power at delta frequencies (0.5 Hz–4 Hz) and low EMG activity; REM sleep, desynchronized EEG with high power at theta frequencies (4 Hz–10 Hz) and low EMG activity. All brain state classification was validated manually.The EEG power spectral density was analyzed with NeuroExplorer . To quantify the sleep quality, we used the EEG power represented by the power spectral density in the different frequency ranges relative to the average value of total power in the same epoch.Sleep deprivation was achieved through gentle touch with a soft brush pen over a period of 1 h or 3 h after acute SDS. Mice were singly housed and gently touched for 1 s–2 s as soon as they displayed signs of drowsiness.3) under low light (~20 lux in the center and ~5 lux in the corners) to minimize anxiety effects. Mice were placed in the center of the arena, then the total locomotor activity in 5 min and the time in the center area were recorded and analyzed using the ANY-maze system .The OFT was used to assess anxiety-related behavior and locomotor activity in an open field arena . In all cases, Animals suffer from social defeat mainly in the active period. However, all previous studies of mice started the social defeat session in the inactive period when the duration of baseline NREM sleep is relatively longer –13. In rFirst, to investigate the effects on sleep of SDS starting in the active period, we analyzed EEG and EMG recordings at baseline  and within 24 h after SDS. When returned to the home cage, the intruder mice showed a robust decrease of time in wakefulness Fig. A. We comP = 0.19, Fig. Next, to define the precise time window of the increased NREM sleep, we analyzed the sleep/wake profile at 15-min resolution during the first 6 h after SDS Fig. A, D, G. Moreover, we compared the first episode of NREM sleep to baseline  using two parameters that indicate sleep quality, EEG power and sleep duration. NREM sleep after SDS showed a significant decrease in EEG power in the delta band Fig. A and a sAlthough the total time of REM sleep in the first 3 h after acute SDS did not significantly increase, it showed a robust increase at one time point Fig. G–H. UnliSeveral studies have demonstrated that sleep loss is closely associated with the occurrence of symptoms of anxiety , 24, 25.Because sleep deprivation is considered to be an anxiogenic factor in humans, we investigated whether acute sleep deprivation with gentle touch for 3 h causes anxiety-like behaviors in normal mice. In the absence of SDS, 3 h of sleep deprivation changed neither locomotion Fig. B, nor anad libitum sleep in their home cage, and sleep-deprived mice. After 1 h or 3 h of ad libitum sleep or sleep deprivation after SDS, each mouse was tested in the OFT and EPM . Supporting the hypothesis that more anxious individuals find it harder to fall asleep, the latency to sleep onset following acute SDS predicted the level of anxiety: mice taking more time to fall asleep were more likely to spend less time in the open arms of the EPM after 3 h of ad libitum sleep; this is interpreted as indicating a high level of anxiety.In addition, we found that the latency to sleep onset reflected the anxiety level after SDS and natural sleep, as it was positively correlated with anxiety-like behaviors (or a negative correlation with time in the open arms) (Fig. All the above data suggest the essential role of sleep in the relief of stress and modulation of anxiety.ad libitum returned the anxiety level to normal.In this study, we focused on the anxiolytic effect of increased NREM sleep in mice experiencing acute social defeat. Our results showed that acute stress in the active period increased the duration of subsequent NREM and REM sleep. In particular, NREM sleep but not REM sleep significantly increased within the first 3 h after social defeat. Deprivation of this increased NREM sleep maintained a high level of anxiety in socially defeated mice, but sleeping The strong increase in sleep after acute social defeat indicated that sleep may function to offset the mental load imposed on the nervous system during wakefulness. In fact, social conflict in rodents enhances the amount and intensity of subsequent sleep following stress-induced insomnia ; sleep rUsually, the anxious state induced by acute social defeat does not last long in naive mice. Mice rapidly recover to a normal anxiety level, which prevents the pathogenesis of anxiety disorders. However, the mechanism by which individuals spontaneously recover from stress is still not well studied . Here, bIn laboratory studies, anxiety or depression is often modeled with chronic social defeat for 7 days or 10 days. In chronic social defeat, the presence of sensory contact with a CD-1 mouse may change the subsequent sleep profile, which would have profound effects on recovery from anxiety. During the sensory contact period, the intruder mouse remains in the aggressor’s home cage but separated from it by a baffle that allows visual, auditory, and olfactory interaction with the aggressive CD-1 mouse , 27, 28.Here, we used sleep deprivation to study the function of increased NREM sleep in anxiety. Indeed, chronic sleep deprivation is reported to be closely associated with anxiety and negative emotion in humans , 29–31. In our results, REM sleep-rebound occurred later than the increased NREM sleep. REM sleep is an adaptive response to stressful situations  and playHowever, the mechanism underlying the anxiolytic effect of sleep is still unclear. The reduction of anxiety-like behaviors is unlikely to be related to memory loss because NREM sleep contributes to memory consolidation –38. We s


Anterior cruciate ligament reconstruction surgery is one of the most common orthopedic procedures. One of the main factors that influence the outcome is regaining strength in the postoperative phase. Because anterior cruciate ligament reconstruction surgeries are often performed in young patients, we combined the concept of prehabilitation with an app-based serious gaming approach to improve maximal strength postoperatively.Our objective was to conduct a prospective randomized trial to evaluate whether an app-based active muscle training program (GenuSport Knee Trainer) can improve postoperative strength by starting rehabilitation immediately after primary anterior cruciate ligament reconstruction surgery.We designed a pilot study in which we randomly assigned patients receiving primary anterior cruciate ligament reconstruction to either the serious gaming training (intervention) group or a conventional rehabilitation (control) group. Except for the serious gaming-based training, both groups followed the same postoperative treatment protocol. Outcome parameters were absolute and relative change in maximal strength, as well as the International Knee Documentation Committee Subjective Knee evaluation form, Knee Injury and Osteoarthritis Outcome Score, and Lysholm Knee Score.P=.02). Similarly to the absolute increase, the relative change in maximum strength was relevantly higher in the exergaming group. The mean relative change in maximum strength was 1.7 (SD 1.17) in the intervention group and 1 (SD 0.13) in the control group. No adverse events or problems were reported during the study period.In total 26 patients agreed to participate . We noted a difference in absolute maximum strength between the exergaming intervention and the control groups. Mean maximum strength preoperatively was 155.1 (SD 79.2) N in the intervention group (n=14) and 157.0 (SD 40.8) N in the control group (n=11). Postoperative mean maximum strength was 212.8 (SD 78.5) N in the intervention group and 154.5 (SD 27.1) N in the control group. Mean absolute change in maximum strength was 57.7 (SD 95.2) N in the intervention group and –4.8 (22.2) N in the control group. The analysis of covariance model with absolute change as the dependent variable and treatment group and baseline maximum strength as covariates showed a relevant difference in relative change between treatment groups (intervention – control) of 59.7 N (95% CI 10.1-109.3; Implementation of an app-based active muscle training program in the early postoperative therapy scheme was associated with an improvement in maximal strength. Therefore, we considered the use of GenuSport training after anterior cruciate ligament reconstruction to be a helpful complement to rehabilitation after anterior cruciate ligament reconstruction surgery to improve strength in the early postoperative phase. To our knowledge this was the first study to analyze immediate postoperative serious gaming-based training with the GenuSport device based on strength improvement. The rupture of the anterior cruciate ligament (ACL) is one of the most common ligament sports injuries of the knee joint. The number of cruciate ligament operations performed is quite high and has increased continuously over the last years. Because ACL ruptures often occur during sport activities, a relevant number patients are under the age of 40 years .Over the past years, many changes have been achieved, especially in surgical reconstruction techniques. Nonetheless, especially in the last decade, studies have also focused on rehabilitation after ACL reconstruction and on possible strategies to improve outcomes. It is well known that, in addition to several other preoperative and postoperative factors, insufficient rehabilitation leads to a poor outcome in terms of restricted range of motion, strength, and overall knee function, resulting in arthrofibrosis -4. Due tThe tremendous importance of early rehabilitation in all fields of medicine, for example, after neurological disorders  or surgeries in general, has been understood over the last decades. That is why new concepts have been implemented in the perioperative and postoperative phases. The first concept of enhanced recovery after surgery, consisting mostly of protocols in which patients were guided by physiotherapists, already led to a clinically significant improvement in rehabilitation -8. New cOne problem of most available feedback-controlled training systems is that the exercises mainly focus on range of motion, and training is only possible with an acceptable range of motion. Therefore, we previously tested a device in rehabilitation after total knee arthroplasty focusing on feedback-controlled active muscle training uncoupled from range of motion. The GenuSport Knee Trainer improved the outcome after total knee arthroplasty especially in the immediate postoperative phase . AdditioWe postulated that training with our device in the initial postoperative phase after ACL reconstruction surgery would improve the overall postoperative outcome, thereby helping the predominantly young patients on their return to normal life and especially to an early return to sports. In comparison with other studies that mainly focused on improved range of motion rather than strength improvement, our device can be used directly after surgery . This alTo our knowledge, no studies have analyzed the effect of muscle strength–based training immediately after ACL surgery. We developed this randomized controlled trial to address this lack.In this prospective randomized controlled trial, we recruited patients awaiting primary ACL reconstruction surgery at a single tertiary health care center between April 2016 and February 2018. The trial is registered; We previously described the Knee Trainer device -19. It cDue to the nature of this study, blinding of patients was not possible.Surgeries were performed by the same senior physician. In all patients, hamstring grafts were used. The fixation technique included femoral and tibial screw fixation , as well as extracortical fixation .We analyzed all patients before and 6 weeks after surgery. We measured function using the International Knee Documentation Committee Subjective Knee (IKDC) evaluation form, Lysholm Knee Score, Tegner activity scale, Knee Injury and Osteoarthritis Outcome Score (KOOS), the maximum strength, and a visual analog scale. Additionally, we measured the femoral diameter 10 cm and 20 cm above the joint line.We analyzed change in maximum strength as both the absolute difference between the week-6 and presurgery values (6 weeks – presurgery) and as the relative change (6 weeks / presurgery). We compared the change in maximum strength on both scales between the intervention and control groups using an analysis of covariance (ANCOVA) model with change in maximum strength as the dependent variable and presurgical maximum strength and treatment group as the independent variables.We performed statistical analysis using IBM SPSS Statistics version 25 (IBM Corporation).In total 26 patients agreed to participate (14 patients in the exergaming group and 12 patients in the control group). One patient of the control group was lost to follow-up; hence, we analyzed 25 patients in total (14 intervention patients vs 11 control group patients) .We saw high compliance rates within the intervention group. None of the patients needed more than 1 instruction to use the knee trainer. Of the 14 patients in this group, 11 (79%) trained with the device regularly (around 17 times a week), whereas 3 patients (20%) did not comply with the training frequency of at least 7 times a week.P=.02)  and the control group (n=11). Postoperative mean maximum strength was 212.8 (SD 78.5) N in the intervention group (n=14) and 154.5 (SD 27.1) N in the control group (n=11). Mean absolute change in maximum strength was 57.7 (SD 95.2) N in the intervention group and –4.8 (SD 22.2) N in the control group. The ANCOVA model with absolute change as the dependent variable and treatment group and baseline maximum strength as covariates showed a relevant difference in absolute change between treatment groups  of 59.7 N  .P=.03) (Relative change in maximum strength was higher in the exergaming group. Mean relative change in maximum strength was 1.7 (SD 1.17) in the intervention group and 1 (SD 0.13) in the control group. The ANCOVA model with relative change as the dependent variable and treatment group and baseline maximum strength as covariates showed a relevant difference in relative change between treatment groups  of 0.67  .A subgroup analysis of patients who trained more than 2 times a day showed no difference in absolute and relative changes in maximum strength, but we saw a clear trend to higher strength levels in the group of patients that trained more than 2 times a day.Changes in clinical scores  were not different between the 2 study groups .Neither group reported adverse events during the study period. In particular, there were no readmissions to the hospital, reoperation for any reason, infections, or falls.Following the progress made in information and communication technology, new technology-based rehabilitation strategies have been incorporated into various medical fields. In this context, new protocols for rehabilitation have been developed. These serious games for functional rehabilitation are considered to improve the whole rehabilitation process. Notably, in most published studies, patient compliance has been very high. This indicates a high motivation for training with exergaming devices. These results for serious games have been reported in all fields of medicine  -25. AlthAll of the patients allocated to the exergaming group completed the entire follow-up protocol. In the exergaming intervention group, about 80% showed good compliance, meaning they trained at least once a day, and none of these patients needed face-to-face contact during the 3-week training period. This indicates that the device design is user-friendly and that people from different age groups can use the device autonomously after adequate initial instruction. Ideally, instruction would start before surgery. We think that there is also potential for using the device in the preoperative phase to improve preoperative strength, which is known to positively influence the postoperative outcome ,27.We saw a slight trend in the frequency of daily training decreasing over the 3 weeks. We think that this might have been because the gameplay of the 2 game modes that were provided did not develop over time in difficulty or the main game principle. To achieve the goal of implementing serious gaming into the rehabilitation of musculoskeletal disorders, existing systems must be evaluated and further developed . To thisThe most important problem in feedback training strategies is that nearly all of them require an adequate range of motion. Therefore, therapy cannot start immediately after surgery because the patient is not able to properly move his or her legs. We designed our study to assess whether it is possible to positively influence the immediate postoperative phase by following a novel exergaming-based training protocol based on strength training rather than on range of motion as in other protocols. Other recently published studies of feedback-controlled training after total knee arthroplasty started their programs not before day 7 . ResultsBecause serious gaming has already been incorporated into other fields of medicine with good results, musculoskeletal surgeons should also implement these promising strategies both before surgery and into rehabilitation after common procedures, as well as in conservative orthopedics. The good results in different age groups suggest that serious gaming is applicable in nearly all periods of life -31.The main limitations of our study were a comparatively small patient population and a short follow-up period of 6 weeks. This is due to the fact that our approach to the immediate postoperative strength-based training has not been implemented in postoperative protocols. Hence, we conducted this pilot study to evaluate the effect of exergaming-based training on the early postoperative phase as a basis for further studies.To our knowledge, this was the first study that successfully implemented a serious gaming-based strength training device into early rehabilitation after ACL surgery. The results are promising; hence, we think that this technology has high potential in this area of rehabilitation, as well as in prehabilitation of musculoskeletal disorders. Further studies are needed to test these concepts and results. If the results of these further studies are as promising as the results of our study, we think that this might be a big step toward implementing such training strategies into routine clinical protocols, especially since the positive aspects of serious gaming are seen in all age cohorts.


To create an intelligent surface region of interests (ROI) 3D quantitative inspection strategy a reality in the continuous casting (CC) production line, an improved 3D laser image scanning system (3D-LDS) was established based on binocular imaging and deep-learning techniques. In 3D-LDS, firstly, to meet the requirements of the industrial application, the CCD laser image scanning method was optimized in high-temperature experiments and secondly, we proposed a novel region proposal method based on 3D ROI initial depth location for effectively suppressing redundant candidate bounding boxes generated by pseudo-defects in a real-time inspection process. Thirdly, a novel two-step defects inspection strategy was presented by devising a fusion deep CNN model which combined fully connected networks (for defects classification/recognition) and fully convolutional networks (for defects delineation). The 3D-LDS’ dichotomous inspection method of defects classification and delineation processes are helpful in understanding and addressing challenges for defects inspection in CC product surfaces. The applicability of the presented methods is mainly tied to the surface quality inspection for slab, strip and billet products. In recent years, with the advent of the industrial 4.0 enterprises undergoing transformation and upgrading manufacturing processes, continuous casting (CC) as a main solidification process for molten steel has been widely popularized to produce metal semi-finished products . In the In the CC production line, with the improvement of quality requirements, the defect depth has become a significant factor, which, especially for the CC slab, sometimes may cause potential security problems. In other words, some defects can be ignored or repaired by the follow-up finishing process if the depth of the defects does not exceed a certain value. Furthermore, conventional optical imaging 2D inspection methods are susceptible to high-temperature radiation interference. In this work, we refer to the entire defects inspection process as two separate steps: recognition and delineation, and based on our previous work in , a novelIn image-based ROI inspection methods, it is a prerequisite for the imaging sensor to be able to capture objects informatively and adjust imaging parameters adaptively as the peripheral environment changes. Therefore, the 3D-LDS as a structured light assistant active imaging system needs a laser stripe with a maximal color contrast and the most homogeneous gray-level. Namely, the imaging sensor should be set to an appropriate optical integral time (OIT) and focus status. When it comes to a rigid system architecture, the focus status can be fixed as the imaging distance and imaging depth of field (DOF) are constants. However, the automatic OIT controlling method needs to be focused on if imaging sensor works in an unstable high-temperature radiation environment. According to the Planck theorem , we tookOn the basis of the light radiation principle, we presented an improved method to determine threshold  follows :(1)E0={μb=∑To implement the deep learning 3D inspection method and create a reliable detection system to meet the special requirements, we devised an improved experimental system based on our previous research. In the system, the 3D images pixels (12-bit) are indirectly mapped from the calibrated laser triangulation strategies (the metric is millimeter). Therefore, the image ROI was reconstructed by converting the 3D distance point cloud of the object surface. From the experiments in N individual neurons , where, loss as formulated as below:1*T that only the value of the position corresponding to the real label is equal to 1. Therefore, this formula actually has a simpler form when cross entry is equal to softmax loss while the input softmax. In our work, we set the activation function as softmax in dense layer. Based on the above discussion, we can define the function of the optimization to minimize (or maximize) the loss function andandBased on the softmax function we can introduce the softmax  include  batch gr include . Adam pr follows :(12)mt=βDue to the all-pervading oxide scales on CC products surface have similar characteristics with real defects, especially in 2D images while it is processed by imaging processing algorithms. We call it pseudo defects interference in inspection process as presented in function to obtain different output. However, the final binary image will be segmented by a fixed threshold.(i)In the training process, the RI problem will make CNN-DL model training more challenging to converge and become time consuming because of the unbalance of positive and negative pixel samples.(ii)In the testing process, RI defects always get undesirable segmentation results by automatic strategies due to the inaccurate positioning by traditional bounding box.In the 3D-LDS defect inspection process, there is a sensitive parameter: the radius of the candidate bounding box(BBox-R), which determines the size of the ROI relative to the size of BBox. Generally, in order to ensure the candidate BBox includes the ROI accurately. We can set a relatively large radius to locate the ROI. However, this will lead to regional imbalances (RI) and consequently, bring about two main issues, especially in full convolutional networks training and the testing process:mean computing way instead of the max method to prevent isolated point noise interference:We also used the FP and FN to give us an overall understanding for the predicted results. Because both of the FP and FN are errors in data reporting in which a test result improperly indicates presence of a condition. In general, we will get under segmentation results if the FP is greater than FN and and vice versa. Meanwhile, we utilized M-HD to check the predicted boundary as it is sensitive to it. However, we use the In allusion to the running time, we tested on the computer with two GPU cards: GEforce GTX 1080 and GEforce RTX 2080Ti, the 2080Ti was used to do the delineation and tested on the maximum BBox(320 × 320). It can perform 15 image segmentation tasks per second that meets the CC production online detection process. With regard to the image scanning speed, we tested image size: 1200 × 600 (the selected CCD cameras is 14fps in full resolution: 2592 × 1944). The system can finish 45fps laser scanning because only laser ROI will be processed in the image. Therefore, the casting speed should be less than 0.8 m/min if the scanning spacing is 0.3 mm. Actually, in real application, the high-performance image workstation or multi-machine distributed processing is preferred. The quantitative experimental results are given in 1)An optimal CCD laser image scanning method was designed in different high-temperature experiments.2)In allusion to defects precise recognition, we proposed a novel region proposal method based on the 3D ROI initial location that can effectively suppress redundant candidate bounding boxes generated by pseudo-defects in a real-time recognition process.3)To improve the inspection accuracy, a deep CNN architecture combined fully connected networks (for defects classification) and fully convolutional network (for defects delineation) was proposed to robustly make the whole inspection methodology defined as a two-step process.4)The applicability of the presented methods is mainly tied to the surface quality inspection for slab, strip and billet products etcetera. Systematically, A 3D-LDS deep learning system is devised for CC products surface quality evaluation that allows an automatic way of AI algorithms to be applied to the MV inspection field in modern industries.In this paper, an improved binocular vision-based 3D laser image scanning deep-learning system (3D-LDS) was established for CC products surface evaluation. The main work is as below: Future work: Based on the experimental analysis, it is found that the optimization of network architecture is a long-term job. There is no unified network model for different detection tasks and targets. Therefore, it is essential to conduct field experimental studies to improve and construct a more robust network architecture especially for the defects classification network. The aim is to solve the common over-fitting problem of current networks and to reduce the dependence on data source quality in model training process. Furthermore, the improvement method of optimization algorithm for deep CNN model training should be further studied through the deep neural network mechanism research in the specific application context. In the following work, we will carry out field experiments and application research in the continuous casting production line.


The anatomical complexity of the oropharynx and the difficulty in reaching its distal portion have always conditioned the surgical accessibility.Robotic surgery represents an excellent alternative in the treatment of cervico-facial oncological diseases.This series comprises all patients managed for head and neck cancer by Trans Oral Robotic Surgery TORS.The staging assessment, including neck ultrasound and total body PET/CT scan, was performed in each patient according to the TNM classification.All charts were recorded with the following data: name and surname, age, gender, date of surgery intra or post-operative hemorragia, tumor site, histology, TNM stage, robot set-up time, tumor resection time, whether or not tracheotomy was performed, whether or not neck dissection was performed, insertion of a nasogastric tube or gastrostomy, time to resumption of oral feeding, surgical margins, mean length of hospital stay, adjuvant treatment and follow-up.From February 2013 to February 2018, TORS was performed in 67 consecutive patients affected by head and neck tumours.We divided, our sample, in 3 subsites: supraglottic larynx, parapharyngeal space and oropharynx.Pathology reports confimed malignancy in 44 cases: 8 cases lymphomas, 36 cases of Squamous cell carcinoma (SCC), 5 cases of benign salivary glands tumors and 18 miscellaneous cases. Neck dissection was performed in 12 cases.Tracheotomy was perfomed in 3/67 cases for respiratory failures. A nasogastric tube was inserted at the end of the surgical procedure in 21 patients. The mean length of hospital stay was 10 days .Major complications included post-operative bleeding in 3 patients, 1 exitus for massive bleeding 20 days post-surgery and 1 respiratory failure treated with tracheotomy and monitoring in the Intensive Care Unit (ICU) for 3 days.Robotic surgery has been considered a valid alternative to traditional open treatment in many specializations with the advantages of an endoscopic procedure, with the same oncological and functional results and with fewer complications. The advantages of this type of surgical technique have been discussed, it is mandatory to focus on the indications and contraindications. The anatomical complexity of the oropharynx and the difficulty in reaching its distal portion have always conditioned the surgical accessibility, limiting the traditional transoral resection possibilities to the upper oropharynx .For decades the surgical gold standard was the trans-mandibular approach, but this was burdened by important morbidities and high complication rates.Robotic assisted surgery was first described in 1976 when NASA began to study a method to assist astronauts in orbit by using a technology that allowed telepresence surgery  and 42.3[± 21] minutes, respective.One case was converted to a transoral blunt instrument and finger approach to complete the caudal dissection of the mass.Frozen section examination of the operative specimens was performed in all patients to study the surgical margins.2 patients received adjuvant radiotherapy (RT), 19 patients were treated with chemoradiotherapy (CT + RT), 1 patient was treated with chemotherapy (CT) this group underwent a TORS procedure in advanced local diseases for reduction and debulking purpose; finally and 1 patient was treated with a combination of RT+ surgery on N and CT.Post-operative pain management 7 days after surgery was mainly performed with NSAIDs or paracetamol (75%) or elastomeric pump infusion (25%) (ketorolac + tramadol + ranitidine).The follow-up was performed over a period ranging from 2 months for the patients operated more recently up to 5 years with the following results:regarding the patients with a malignant histology, 36 patients were free of disease (no evidence of disease NED), 3 patients had died of disease (DOD), 6 patients are currently receiving combined radio / chemo treatment  and 1 patient at the last control showed the presence of a second neoplasm in another site (breast carcinoma), with a disease free survival (DFS) of 78.2% and an overall survival (OS) of 93.4%.In the past, most surgery for oral and pharyngeal carcinomas (OPC) was performed through transfacial or transmandibular incisions , 9. AlthTORS, in fact, associated in N+ cases with neck dissection, allows the ablation of the tumor mass with a non-invasive approach that guarantees a 3D view of the surgical field and therefore the possibility of dominating the neoplasm with “safe” margins.The trans-oral approach was first introduced by Huet in 1955 . SteinerFrom the second half of the 2000s, TORS displaced the endoscopic approach, increasing the surgical possibilities, thanks to the overcoming of the technical limits of TLM. This technique offers many advantages: no visible external scars, the 360° motion of the robotic system, three-dimensional high definition visualization, and reduction of hand tremors allow good manipulation of the tissues, improved visualized surgical field, and a shorter postoperative length of hospital stay , 10.Another fundamental concept is “de-intensified treatment”, that represents the reduction of chemotherapeutic drug doses and of the radiotherapy field of action.There are two ongoing ECOG 3311 studies  and PATHOS  positive oropharyngeal cancer) using information about unfavorable prognostic factors, obtained from histological examination, to stratify patients according to risk and thus propose intensifying therapies where possible –25.In 2009, the US Food and Drug Administration approved the use of the da Vinci Surgical System for transoral otolaryngology. Previously, the treatment of OPC had been reserved for a combined radio and chemotherapeutic approach with good results in terms of survival, but linked to acute and late toxicity. Similiar complications were connected with trans-mandibular approaches that guaranteed a good resection with important deficits for the patient and irreversible changes in the quality of life (QOL).These considerations are the basis of the concept of de-intensified treatment that allows us to take advantage of TORS and therefore a minimally invasive surgery with positive results in terms of survival , 26.Optimizing the quality of life of these patients, reducing the toxicity of the radiation dose to be administered, without compromising the possibilities of treatment, has become imperative , 26.Today it is therefore possible to reduce the radio-chemotherapy treatments in “intensity”, with a consequent decrease in morbidity.In support of this thesis, TORS is an emerging treatment option in order to modulate the adjuvant therapy –17 avoidThe TORS data are encouraging, the parameters studied being the surgical feasibility ie the analysis of the set-up time and operational time and oncological and functional outcomes  –30.Once the advantages of this type of surgical technique have been discussed, it is mandatory to focus on the indications and contraindications.Mouth opening must be evaluated to ensure a correct positioning of the robotic arms and exposure of the working space. Trismus (mouth opening of less than 1.5 cm), macroglossia, and mandibular/maxillary defects may represent a major contraindication to performing TORS. Furthermore, much is still debated regarding the predictive markers of contraindication to TORS, and even preoperative cephalometric analysis has been proposed. MRI is reliable in ruling out carotid encasement and bone erosion .Nevertheless, tissue adherences may complicate dissection, and if these are located in the lateral/posterior part of the mass, towards the external carotid, a change of approach to transoral blunt instrument and finger dissection may be required. As suggested by other authors, the intraoperative decision to switch to a transoral blunt dissection may be necessary − or even advisable - in order to guarantee patient safety (minimize the risk of major haemorrhaging from the carotid artery) and tumor capsule integrity in the absence of particular predictive radiographic signs , 31. AltWe analyzed sufficient mouth opening in all patients allowing adequate exposure of the tumour;accurate diagnostic workup for the selection of cases is necessary to the indication of TORS.Furthermore, patients must have transoral access to the oropharynx. T1,T2 oropharyngeal cancers are amenable to resection, above all if they arise from the lateral tongue base, the tonsillar fossa, the lateral pharyngeal wall and the glossopharyngeal sulcus . ParticuRegarding the literature, Weinstein et al. reported 100% locoregional control for T1–3 tonsillar carcinomas in 27 patients . PatientP < 001). Other smaller studies do not suggest any prognostic impact of the status of HPV for T1–2 patients with resectable OPC if treated with an initial TORS approach [Still debated in the literature is the relationship between HPV and TORS and there are still no clear indications about the possible differences in the use of TORS in patients with positive or negative HPV. Ang et al.  highlighapproach .P < 001) and feeding tube use (P < 001) and shorter overall hospital stays. The 2-year recurrence-free survival rate was significantly higher in the TORS group than in the open approach group . In our series only 5 cases out of 67 (7.46%) underwent surgical salvage treatment for local advanced diseases previously treated with CHT and RT.In addition, doubts have been raised regarding the high rate of adjuvant radiotherapy in TORS patients but a study by Weinsten et al.  reportedRegarding the comparison between TORS and open head and neck surgery, a large review of the National Hospital Data Survey database between 1995 and 1997 including 3932 patients in non-robotic head and neck procedures  looked aIn our study we found a major complication rate of 5.97% and a mortality rate of 4.4%.p < .001). The mean durations of feeding by nasogastric tube and hospitalization were shorter for the TORS group (p = .001). There was no significant difference in disease-free survival at 3 years (p = .76). The mean treatment cost was $7124 lower for the TORS group (p = .03). In our group we performed only 3 tracheotomies in 67 procedures (4%).Hammoudi et al. in 2015  comparedFrom our experience it has emerged that the robot allows us to operate in patients with benign tumors, diagnostic lymphomas, miscellaneous and T1-T2-T3 OPC in narrow and anatomically complex spaces, while for the more advanced stages radiant treatment is preferable.In lymphomas cases we had a limph-node fnab indicative for immunoproliferative process but was mandatory histological characterization, moreover the oropharynx localization gave severe dysphagia and in 2 cases oropharynx localization was the only recurrence site.Regarding patients with a malignant histology, 36 patients were free of disease (no evidence of disease NED), 3 patients died of disease (DOD), 6 patients are currently receiving combined radio / chemo treatment  and 1 patient at the last control showed the presence of a second neoplasm in another site (K breast), with a disease free survival (DFS) rate of 78.2% and an overall survival (OS) rate of 93.4% with a mean follow up of 30 months.The most frequent sequela reported by our patients was postoperative pain controlled with paracetamol or with the use of NSAIDs 7 days after surgery in 60% of patients.TORS allows us to perform transversally the same interventions that can be carried out with the classic open procedure, but with the advantages of an endoscopic procedure, with the same oncological and functional results and with fewer complications.It is possible to avoid invasive procedures such as mandibulotomy, reduce the range of radiotherapy (de-intensified treatment), reduce the need to perform a tracheotomy, avoid the onset of problems related to a possible iatrogenic malocclusion, reduce intra and post-operative bleedings, reduce the possibility of infections due to the less extensive surgical site, favor a faster functional recovery by decreasing the number of patients undergoing enteral nutrition in the post-operative period, decrease the presence of visible scarring and shorten hospitalization times.From a literature review it has emerged that the problems of the management of patients undergoing major cancer surgery can be addressed with the choice of TORS so reducing average hospitalization times .With robotic surgery the same results can be achieved but with a lower morbidity .The current trend is to consider robotics for T1-T2 the indication for this surgery are currently being extended to glottic tumours and larger lesions such as some T3 -T4 tumors , 42.We performed a TORS procedure in local advanced diseases for disobstructive and cytoreductive aims and to obtain better and broader histological material for histopathological characterization;this explains the number of positive margins.In conclusions TORS represents a good tool for staging and treating neoplasms of head and neck cancer –46, the


A novel disease poses special challenges for informatics solutions. Biomedical informatics relies for the most part on structured data, which require a preexisting data or knowledge model; however, novel diseases do not have preexisting knowledge models. In an emergent epidemic, language processing can enable rapid conversion of unstructured text to a novel knowledge model. However, although this idea has often been suggested, no opportunity has arisen to actually test it in real time. The current coronavirus disease (COVID-19) pandemic presents such an opportunity.The aim of this study was to evaluate the added value of information from clinical text in response to emergent diseases using natural language processing (NLP).We explored the effects of long-term treatment by calcium channel blockers on the outcomes of COVID-19 infection in patients with high blood pressure during in-patient hospital stays using two sources of information: data available strictly from structured electronic health records (EHRs) and data available through structured EHRs and text mining.In this multicenter study involving 39 hospitals, text mining increased the statistical power sufficiently to change a negative result for an adjusted hazard ratio to a positive one. Compared to the baseline structured data, the number of patients available for inclusion in the study increased by 2.95 times, the amount of available information on medications increased by 7.2 times, and the amount of additional phenotypic information increased by 11.9 times.In our study, use of calcium channel blockers was associated with decreased in-hospital mortality in patients with COVID-19 infection. This finding was obtained by quickly adapting an NLP pipeline to the domain of the novel disease; the adapted pipeline still performed sufficiently to extract useful information. When that information was used to supplement existing structured data, the sample size could be increased sufficiently to see treatment effects that were not previously statistically detectable. Outbreaks of novel diseases can create enormous strain on public health systems. Since the time of Snow's pioneering work  on the eBiomedical text mining, or the use of textual data, in electronic health records (EHRs) has often been proposed as a method for converting unstructured data to the structured data that is needed in public health informatics. One of the advantages of biomedical text mining is that it can be developed rapidly , which cThe data used in this study were obtained from 39 different hospitals in the Paris metropolitan area in the Assistance Publique – Hôpitaux de Paris (AP-HP) system. Focusing on this region of the country and on a large number of hospitals afforded a diversity of patient demographics that would not be available in most other parts of the country. As of May 4, 2020, the Entrepôt de Données de Santé (EDS)-COVID data set contained 84,966 electronic records of suspected or confirmed patients with COVID-19  were applied to the full data set  were extracted via a deep learning approach based on bidirectional encoder representations from transformers (BERT) contextual embeddings  , scores  and physiological measures , were extracted via a list of 60 regular expressions (NLP RegExp).All signs, symptoms, and comorbidities included in the Unified Medical Language System (UMLS)  were extA visual depiction of the pipeline is provided in The NLP medication extraction model was a bidirectional long short-term memory with a conditional random field (BiLSTM-CRF)  layer onThe NLP regular expression for the extraction of specific phenotypes was a set of 60 regular expressions developed manually and iteratively by medical informatics experts and physicians. We evaluated their precision at the sentence level using a random sample of 100 positive sentences for each regular expression. Examples of these expressions can be found in All the terms extracted by the NLP pipeline, regardless of the method, were automatically annotated according to their modality  and experiencer in the text, as described in previous work . The outData supporting this study can be made available on request, on condition that the research project is accepted by the scientific and ethics committee of the AP-HP health data warehouse .The clinical goal of this case study was to evaluate the potential effects of CCBs on in-hospital mortality related to COVID-19 . To achiWe considered a patient as receiving long-term treatment with CCBs  if thereP=.05, and all statistical tests were two-sided. We used R statistical software v.3.6.2 (R Project) with the Survival package.The measured outcome was in-hospital mortality. We used a multivariate Cox proportional hazard model  that wasNLP medication)⁄(structured medication), and the number of phenotypes increased by 15.2 times ((NLP RegExp + NLP UMLS)⁄(ICD-10 codes). Among the 84,966 patients with records present in the EDS-COVID cohort  in all sections. When focusing on the admission and discharge treatment sections, the F1 score was 96.7% . The detailed results are shown in Of the 84,966 total patients, 3965 (4.7%) were included using the NLP pipeline, of which only 1343 (15.9%) could be included if the study were limited to the use of structured data; this increased the number of patients added for the case study increased by 2.95 times . A detaiP=.12). When including NLP data, the aHR became 0.82 (95% CI 0.71-0.94), which represents a statistically significant reduction of the risk of death (P=.005). Similar results can be observed that support an increased risk of mortality with the presence of diabetes and cancer as comorbidities  of 0.83 (95% CI 0.67-1.05) for treatment with CCBs; this result was not statistically significant , radiological signs , comorbidities , and drug history , requiring extraction of information from narrative reports in EHRs.The case study described in this paper shows the possible impact of using information extracted from text in the EHR for COVID-19 research. More precisely, the conclusions of the above study would have been different if information from unstructured fields had been excluded. In our case study, the addition of information from NLP did not dramatically change the hazard ratio from the analyses; however, it allowed us to include more patients and therefore narrowed the CIs and increased the statistical power. Note that the increased statistical power is mainly due to the increase in the number of patients included and the quantity of data available. Further analyses are required to assess the validity of the associations detected here, given that some confounding biases may remain and provoke false positive results. Reproducing the analysis with an external population or performing falsification testing  could he