In vivo, climbing fibers spike continuously, including during movements when parallel fibers are simultaneously conveying sensorimotor information to PCs. Whether parallel fiber activity enhances climbing fiber Ca2+ signaling during motor behaviors is unknown. In mice, we found that inhibitory molecular layer interneurons (MLIs), activated by parallel fibers during practiced movements, suppressed parallel fiber enhancement of climbing fiber Ca2+ signaling in PCs. Similar results were obtained in acute slices for brief parallel fiber stimuli. Interestingly, more prolonged parallel fiber excitation revealed latent supralinear Ca2+ signaling. Therefore, the balance of parallel fiber and MLI input onto PCs regulates concomitant climbing fiber Ca2+ signaling.Motor learning involves neural circuit modifications in the cerebellar cortex, likely through re-weighting of parallel fiber inputs onto Purkinje cells (PCs). Climbing fibers instruct these synaptic modifications when they excite PCs in conjunction with parallel fiber activity, a pairing that enhances climbing fiber-evoked Ca Climbins in PCs  in respos in PCs . Howevers in PCs . Therefos in PCs . The ints in PCs .2+ signals in PC dendrites are enhanced in a supralinear manner (2+ elevation that triggers long-term depression (LTD) at parallel fiber-PC synapses  driving feed-forward inhibition that can attenuate parallel fiber excitatory postsynaptic potentials (EPSPs)  . MLIs ca (EPSPs)  and impa (EPSPs) . Consequ2+ signals in PC dendrites in vivo to determine how these responses are regulated by local circuit activity in the cerebellar cortex of awake behaving mice. During the performance of practiced movements, we found that parallel fiber and climbing fiber co-activity failed to produce supralinear Ca2+ signals. However, disinhibiting the molecular layer through chemogenetic suppression of MLI activity enhanced the amplitude of climbing fiber-evoked Ca2+ signals in PCs, specifically during movements when both parallel fibers and MLIs were activated. Quantitative ex-vivo measurements in PC dendrites confirmed that MLI-mediated feed-forward inhibition limits the ability of parallel fiber excitation to produce supralinearity. Bi-directional optogenetic actuation of MLI activity during parallel fiber stimuli altered associative parallel fiber-climbing fiber Ca2+ signaling, dependent on the level of MLI output. Our results show that climbing fiber Ca2+ signals in PCs are regulated by the counterbalance of MLI-mediated inhibition with parallel fiber-evoked EPSPs.We examined climbing fiber-evoked Ca2+ activity in PC dendrites of lobule Crus II in head-fixed mice conditioned to lick for water from a port when cued by an audible tone  to measure climbing fiber-evoked Cable tone . After pble tone . Climbinpromoter . During sumption . This be Crus II . As with Crus II , averageick rate , indicatick rate .2+ signaling in PCs, we collected well-isolated individual dendritic Ca2+ events from PCs  bursts of firing and that the variance in their amplitudes reflects heterogeneity in the number of action potentials contained in the burst  nor did it affect the amplitude of events evoked during licking when MLIs are normally activated  was abolished in alternate trials when MLIs were optogenetically activated coincident with the parallel fiber tetanus  in PC dendrites that can facilitate the initiation and propagation of subsequent climbing fiber-evoked Ca2+ spikes into spiny branchlets, thereby enhancing intracellular Ca2+ entry , excitation and inhibition appeared in balance, as determined by the high correspondence of parallel fiber and MLI activity. This likely limited any enhancement of PC Ca2+ events during task performance. Whether feed-forward inhibition can continuously counteract parallel fiber excitation across a range of behavioral conditions is yet to be determined.In our in vivo recordings, the amplitude of climbing fiber-evoked Ca2+ signals by parallel fibers, MLIs can also reduce the amplitude of climbing fiber-evoked Ca2+ transients through direct electrogenic suppression of PC dendritic spiking , evoke graded climbing fiber-evoked Caendrites . Enhanceendrites . In thisasticity , perhapslearning . In addilearning . Increasses MLIs . Future Kit::Cre mice are not pristinely selective for MLIs ; the latter is expected to be relatively less susceptible to alteration by chemogenetic MLI activity suppression. However, strong hM4d-mediated suppression of MLI neurotransmission, apart from spiking and synaptic activation  at the Max Planck Florida Institute for Neuroscience. Heterozygous adult Cre mice  of both As described previously , cranialloxP)rev-RCaMP2, AAV1-CAG-Flex(FRT)rev-GCaMP6f, AAV1-CAG-Flex(loxP)rev-ChR2.HA-2a-hM4d, AAV1-Syn-GCaMP6f . For injections into lobule Crus II, the micropipette generally contained multiple viruses (100–150 nl) in order to avoid repeated penetrations of the same location. Three different injection depths (150–350 µm below the dura) were used to evenly transduce both PCs and MLIs. To transduce granule cells, AAV1-Syn-GCaMP6f was injected 250–350 mm below the dura  was injected into the inferior olive through a surgical opening in the back of the neck with access to the brainstem through the foramen magnum at the following coordinates from lambda: x = 0.3 mm, y = −4.9 mm, z = −4.6 mm at a depth of 3.6 mm using an approach angle of 62°. Total volumes of injections were ~500 nl. All injection rates were ~25 nl/min.Prior to placement of the window, adeno-associated viruses (AAVs) were pressure injected into the brain using beveled glass micropipettes. Viruses included: AAV1-Pcp2.4-GCaMP6f, AAV1-Pcp2.4-FLPo, AAV1-CAG-Flex(FRT)rev-RCaMP2, AAV1-CAG-Flex and monitored daily for health. In the first few sessions, mice were familiarized with the apparatus and head restraint, and were trained to lick from the water delivery port by providing free access to water. After mastery, demonstrated by consistent licking, and judged by the uptake of water under high-speed videography, a tone cue (6 kHz) was added signaling water availability. After many trials , mice learned to lick at the onset of the tone and refrain from licking after fully consuming the dispensed water droplet. Mice typically completed >150 trails until sate. Tone response time was the interval between the end of the tone cue and the first subsequent lick. Licking movement epochs were defined based on whether a lick had been detected within 0.25 s (roughly two lick cycles). Epochs of non-licking were classified based on the absence of licking for >0.75 s.For experiments in mice expressing the engineered receptor hM4d in MLIs, animals received an intraperitoneal injection of CNO prior to start of the task . Control measurements in the absence of CNO were made from the same mice on alternate days (>48 hr between sessions).2+ activity in neurons of the lateral cerebellum. The microscope was purpose built and included resonant scan mirrors , a low power objective , and high-sensitivity PMTs  for both red and green channels producing high-resolution (512 pixels x 512 pixels), high frame rate images (30 frames/s). This rate is faster than the response time of the Ca2+ indicators  and, after opening the chest, were perfused with ice-cold (~4°C) saline through the heart. The cerebellum was then removed by rapid dissection and mounted on an agar block and sectioned in thin slices (200 μm) using a vibraslicer . Sectioning was performed in an ice-cold solution containing (in mM) 87 NaCl, 25 NaHO3, 2.5 KCl, 1.25 NaH2PO4, 7 MgCl2, 0.5 CaCl2, 10 glucose, and 75 sucrose that was continuously bubbled with carbogen gas (95% O2/5% CO2). Once cut, slices were immediately transferred to a holding chamber containing (in mM) 128 NaCl, 26.2 NaHO3, 2.5 KCl, 1 NaH2PO4, 1.5 CaCl2, 1.5 MgCl2 and 11 glucose, maintained at 34°C for 30 min and then at room temperature (~23°C) thereafter. For experiments, acute slices were placed in a recording chamber under a microscope and continuously perfused with an oxygenated saline solution of identical composition to that used for holding after slicing. The concentration of Ca2+ was kept low (1.5 mM) to reflect a more physiological-like condition . For PC recordings, the Ca2+ indicator dye Fluo-5F  as well as the volume indicator Alexa 594  were also included in the pipette solution. Cell-attached recordings from MLIs were achieved by forming a loose seal with the patch electrode (~400 MΩ). This prevented dialyzing the cell and changing the intracellular concentration of Cl- and, hence, the reversal potential for currents generated by GtACR2.Neurons in Crus II were visually targeted for recording using IR contrast imaging with an upright video microscope  and a QIClick CCD Camera . PCs and MLIs were easily distinguished based on their location and morphology. Recording pipettes were pulled from thin-walled borosilicate glass  and filled with a solution containing (in mM) 124 potassium gluconate, 2 KCl, 9 HEPES, 4 MgClWe used a Multiclamp 700B amplifier  for electrophysiological recordings. Signals were filtered online at 10 kHz and digitized at 20 kHz with a Digidata 1440 A-D converter (Molecular Devices). For whole-cell recordings, the membrane potential of PCs was maintained −75 mV using constant current injections whereas MLIs were maintained at −70 mV. Current offset was not used during cell-attached recordings of MLIs, allowing these cells to fire spontaneously under their own control, at an unperturbed resting membrane potential. Pipette capacitance was neutralized online and series resistance adjusted using the bridge balance circuitry of the amplifier. Liquid junctional potentials, calculated to be 10 mV, were corrected offline. Climbing fibers were stimulated using bi-polar glass electrodes placed near the axon hillock of the targeted PC. Brief electrical pulses  were delivered using a stimulation isolation unit . Parallel fibers were also stimulated electrically using an electrode placed in the molecular layer adjacent to the dendritic recording site. The intensity of the stimulus was adjusted to produce PSPs, recorded at the PC soma, of similar amplitude across recordings (0.1–1.0 mV). For conjunctive stimulation experiments, parallel fibers were stimulated in bursts at 100 Hz. Climbing fiber were stimulated 50 ms after the end of parallel fiber tetanus, or at a varying interval where noted. Trials occurred at a relatively low frequency (0.125 Hz). In interleaved trials, either parallel fibers or climbing fibers were stimulated in isolation. This also occurred for trials that included optogenetic actuation of MLIs. Light pulses for optogenetic actuation or inactivation of MLIs activity started 10 ms prior to the beginning of the parallel fiber tetanus and lasted for the duration of the electrical stimulus.2+ activity in PCs, indicators dyes were allowed to dialyze for >30 min before starting recordings. Inclusion of the red volume dye allowed for identification of dendrites and spines for subsequent Ca2+ activity measurements. However, fluorescence in the red channel was not collected during neural activity measurements because of the interference of light used for the optogenetic stimuli. Ca2+ transients were recorded in PC spines using line scans (500 Hz). The PMT used to collect green Ca2+-indicator fluorescence was shuttered during blue-light optogenetic stimuli to prevent damage. Ca2+ activity was therefore not measured during this period. To facilitate comparison between conditions, a comparable region of the control response was blanked.For 2pLSM imaging in slices, we used a commercial scan head  fitted on top of an upright microscope . The scan head directed laser light (λ = 810 nm) from a mode-locking Ti:sapphire laser  through a scan lens and pair of galvanometer mirrors (Cambridge Technologies) onto the back aperture of a high-power objective . To image CaloxP)rev-GtACR2.eYFP (prepared by ViGene) and AAV5-EF1α-Flex(loxP)rev-bReachES-TS-YFP (University of North Carolina) were injected into the lateral cerebellum of Kit::Cre mice using a surgical procedure identical to that described above except that the size of the craniotomy was reduced (~0.5 mm diameter). Acute slices were prepared from these mice 7 to 14 days after surgery. GtACR2 and bReaChES were activated using blue and amber light, respectively, delivered from separate LEDs . The emission of the LEDS was combined with a dichroic  and directed, unfiltered (λ = 461 ± 20 nm and λ = 596 ± 16 nm), into the back epi-port of the microscope. This light was combined into the 2P excitation pathway using a second dichroic . LEDs were modulated by separate current controllers  using digital commands out of the A-D converter and under computer control from the electrophysiology software .For our slice experiments, AAV1-EF1α-Flex and the chest cavity opened, exposing the heart. The heart was accessed by a needle and the animal was perfused at 2 ml/min with a 0.1 M phosphate-buffered (PB) solution followed by paraformaldehyde (4% by volume in PB) until the perfusate exiting an opening from the pulmonary artery ran clear. The cerebellum was removed by dissection and sliced into 80 µm sections in cold PB. When necessary, HA immunostaining was used to confirm hM4d expression. Samples were first incubated with anti-HA antibody , followed by an Alexa 633 secondary antibody . DAPI  counterstaining was used to identify cell locations in some cases. Images were collected on a confocal microscope  using 488 nm excitation and 493–598 nm emission for GCaMP6f, 633 nm excitation and 638–747 nm emission for Alexa 633, 405 nm excitation and 410–507 nm emission for DAPI, and 514 nm excitation and 519–620 nm emission for YFP.Transgene expression was confirmed by visual inspection of tissue from paraformaldehyde-perfused mice. Following the completion of Ca2+ activity in neurons expressing genetically encoded Ca2+ indicators were aligned using a least-squares algorithm. For dual-color imaging, the translation coordinates from PC images were also used to co-register the corresponding MLI images. Individual PC dendrites or individual climbing fibers were segmented using an independent component analysis algorithm . For overlapping responses, this baseline period was during the decay of preceding events. For trial-averaged PC Ca2+ activity measurements, we calculated ΔF/F for all PC dendrite ROIs using the smallest GCaMP6f-fluorescence values obtained during recordings as the baseline. The average of responses in control was subtracted from that measured in CNO for each mouse before generating an overall average. In bouton measurements from parallel fibers, ΔF/F values were corrected by subtracting the neuropil signal from an area immediately adjacent to each fiber. The MLI-dependent movement (MDM) ratio was defined as the following equation:For in vivo measurements of Ca2+ activity. This was used to generate a ROC curve. The percent of pixels showing a CNO effect was estimated from the area under the ROC curve  from a single dendrite then comparing the amplitudes for each identified event occurring simultaneously at each location. Similar to that of previous work . The peak climbing fiber-evoked Ca2+ transient was determined from an exponential fit of the fluorescence decay immediately following the electrical stimulus.For ex vivo CaAll image analysis was performed with Matlab  or ImageJ (NIH). AxoGraph (Axograph) was used to analyze electrophysiological data. Additional calculations and plotting was performed with Excel  and Prism . In figures, error bars indicate SEM. For data contained within the figure set where individual measurements are not already shown please see  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.2+ signaling in Purkinje cell dendrites during movement" for consideration by eLife. Your article has been reviewed by three peer reviewers, including Jennifer L Raymond as the Reviewing Editor and Reviewer #3, and the evaluation has been overseen by Eve Marder as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Martijn Schonewille (Reviewer #1).Thank you for submitting your article "Inhibition gates instructional CaThe reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:This manuscript by Gaffield and colleagues described an elegant series of experiments investigating the effects of excitatory and inhibitory synaptic inputs to the Purkinje cells climbing fiber-evoked calcium signals. Historically, the climbing fibers were thought to elicit a reliable, all-or-none (binary) calcium response in Purkinje cells, which triggered associative LTD in coactive parallel fibers. However, a number of recent in vitro and in vivo studies have demonstrated that the climbing fiber-elicited calcium response is graded, and that variations in the amplitude of the calcium response can modify the probability that associative plasticity is induced. The current manuscript extends this work by identifying inhibition from the molecular layer interneurons (MLIs) as a signal that can regulate the calcium response elicited by the climbing fibers.The authors employ a powerful combination of complementary in vivo and in vitro calcium imaging, optogenetic and pharmacogenetic manipulation, and slice physiology. They show that the amplitude of calcium transients during a well-learned behavior (licking), which increases both MLI activity and the rate of these transients , is similar to calcium transients in the absence of movement. Chemogenetic inactivation of MLIs, however, unmasks a widespread, movement-specific increase in transient amplitude . This increase appears to be postsynaptic, as direct imaging of climbing fibers fails to reveal an influence of the ligand on presynaptic calcium preceding or during movement . Optogenetic suppression of MLIs in acute slices enhances parallel fiber (PF) postsynaptic potentials at the Purkinje cell soma and induces a supralinear dendritic calcium response to paired parallel and climbing fiber stimulation. Finally, the authors demonstrate that long trains of PF stimulation can induce a similar supralinear calcium response, but that this interaction is erased by optogenetic activation of MLIs.The work is of high potential impact. The results extend our understanding of events that can gate or modulate the climbing fiber's impact on their Purkinje cell targets, which is thought to provide instructive signals that guide the induction of cerebellum-dependent learning. The experiments are well-designed, the claims are clearly explained, and the data support the claims. However, the reviewers agreed that some issues should be addressed by the authors.Essential revisions:1) Because long trains of PF stimulation can induce a supralinear PF+CF effect on calcium, the authors should rule out the possibility that pharmo- and opto-genetic perturbations drives an increase in PF activity . The most direct way to achieve this would be to perform calcium imaging in granule cells and check that activity is similar between treatment and sham conditions. It may also be helpful to provide a more thorough analysis of the effects of the MLI manipulations on behavior. Is enhanced calcium still observed with CNO if the analysis is limited to CNO and control trials with the behavior as closely matched as possible ?2) More thorough analysis of the data, and more complete presentation of the results are needed throughout the manuscript.a) In the analysis of the in vivo calcium events, the authors focus on the unitary calcium responses, and exclude the multipeaked responses from most pf the analyses. The description of how the unitary events were identified is scanty . The authors should provide more information about how they did the analysis and provide an analysis of the robustness of the results to their specific choices for the analysis. What does the distribution of all (single and multipeaked) calcium event amplitudes look like? What is the distribution of single, double, triple peaked events? Does the latter differ during cued licking behavior vs. spontaneous? The example trace in Figure 1C suggests there is surprisingly large variation in the size of unitary events (a ~3-fold difference in the peak with the green check mark vs. the first peak in the multiple event)-this makes me wonder how many of the unitary events selected for analysis are actually composed of two smaller calcium events occurring close together in time. What is the temporal resolution for discriminating one vs. two closely spaced events? A more careful consideration of how the choice of analysis could affect the results and the interpretation of the results is needed.2+ signaling after MLI disinhibition. If I understand correctly the figure also indicates that the other ~37% has a substantial decrease of the Ca2+. This should be addressed, also in relation to the chance of random changes in Ca2+ signaling . Similarly, the remark about the 'arbor-wide' influence should be re-worded to more careful terms.b) The authors show in Figure 3C that ~63% of the dendritic area shows enhanced Cac) For Figure 5D and Figure 6, it would be very helpful if the evoked PF and CF transients were also shown individually, before the summed transient is shown. Also, please state clearly if the summed response for the suppressed inhibition is based on the PF with or without inhibition.d) Figure 6G and associated text suggest that the MLIs exert a graded effect on the climbing fiber-elicited calcium response. Is there truly a graded effect on amplitude, or might it be an effect on the probability of an additive vs. supralinear event of a more uniform amplitude? Additional analysis could distinguish these two equally interesting possibilities.e) The point of Figure 1 is that the amplitude of the calcium events is the same during movement or no movement. However, in Figure 2F, this does not seem to be the case for the hM4d animals, even in the absence of CNO. If the calcium sensing is nonlinear, this could potentially influence what is measured with CNO. This possibility should be considered.f) There are some places where individual examples are shown, but no group data, for example, Figure 4E.g) There are some places where results are presented without statistics. For example, is there is a significant difference between movement vs. no movement and between CNO vs. control in Figure 3FG? Also, the error bars on the CNO-control PC data for simultaneous PC/MLI imaging  are very large. Please provide the same statistical analyses for these data as were used for the data in Figure 2F to justify the claim that the difference is positive. Also, please provide the same analysis for the climbing fiber imaging data in Figure 4G, where the difference appears to be negative, but is asserted to not differ from zero.h) To support the claim that peak PC ΔF/F does not co-vary with MLI activity , please plot compute the correlation between the peak amplitude and MLI activity for all dendrites and complex spike event, and display a scatterplot of these values.i) A video of mouse behavior and two-color imaging  would be useful.j) Figure 5D shows that MLI inactivation induces a supralinear interaction between PF and CF stimulation on dendritic calcium. Is the same true of the somatic membrane potential? Please overlay plots of the PF+CF and PF+CF (sum) for control and light conditions  in Figure 5F, and make a similar plot for the ephys data in the long pulse train experiments .k) Was there any correlation between calcium transient amplitude and behavior? For instance, were transients larger on trials with longer or mistimed licking bouts? Similarly, did the correlation between PC calcium and MLI calcium change with any aspect of behavior? Although the authors have addressed some of these questions in previous work, it would be helpful to have this information for the current manipulations and data.l) In general, the figure legends and text describing the figures should be edited to be more precise and make it easier for the reader to understand what is in the figure. For example, in Figure 2G and associated text, I am guessing that the "totality of all calcium responses" includes the multipeaked as well as the unitary responses, but after considerable effort, I am still not sure. Another example, it is not clear whether the averaged calcium activity in Figure 1F is from one mouse or all mice. Please carefully review all figure legends to make sure that the reader is provided with sufficient information about what is in each figure panel.m) Please provide time units for the event probability plots to allow comparison across figures, and with previously published climbing fiber firing rates. I would expect the probabilities in Figure 1E and Figure 4E to be similar, but they differ by twofold. How does this compare with the typical climbing fiber rate of 1 Hz?3) The current results would have more impact if more effectively framed in the context of what is already known about variations in climbing fiber-triggered responses in the Purkinje cells, how the current work extends what is known, the conclusions that can be drawn, and any caveats.2+ signaling" in the Title and last sentence of the Abstract and "circuit modifications" in the first sentence of the Abstract), given that plasticity is not tested in the in vitro or in vivo experiments.a) Previous work has associated the calcium transients with plasticity, however, the reviewers thought there was too much emphasis on plasticity  Najafi et al., have reported that climbing fiber-associated calcium responses are enhanced when they occur during a learning task as compared with the spontaneous calcium events, which are presumably due to spontaneous climbing fiber spiking. In contrast, the current results indicate that the calcium responses during the well-learned lick task are the same as the spontaneous calcium events. The authors cite Najafi, but don't directly compare the two results so that the reader can effectively appreciate the new finding that the calcium responses are not always enhanced in the behavioral context. This would better frame the current Results section than the current statement in the Introduction "whether CF Ca2+ signal in Purkinje cells during the movement in the lick task is needed. From the example mouse data in Figure 1B, it is difficult to tell how much of the behavioral response and neural activity is learned (a conditioned response) versus an unconditioned response to the water — it looks like the majority of the neural and behavioral response could be unconditioned. Which components of the lick task are cerebellum dependent-acquisition of learning? expression of the condition response? performance of unconditioned licks? Discussion of these issues would help to clarify the potential role of the calicum transients in the behavior.c) More discussion of the cued licking task, and the role of the Cad) In Figure 5 and Figure 6, might the lack of supralinear calcium responses result from the use of a suboptimal parallel fiber-climbing fiber pairing interval?2+ signaling in Purkinje cell dendrites during practiced movements" for consideration by eLife. Your article has been reviewed by three peer reviewers, including Jennifer L Raymond as the Reviewing Editor and Reviewer #3, and the evaluation has been overseen by Eve Marder as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Martijn Schonewille (Reviewer #1).Thank you for submitting your article "Inhibition gates supralinear CaThe reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:This manuscript provides evidence that the molecular layer interneurons in the cerebellar cortex modulate the climbing fiber-triggered calcium responses in Purkinje cells. Historically, the climbing fibers were thought to elicit a reliable, all-or-none (binary) calcium response in Purkinje cells, which triggered associative LTD in coactive parallel fibers. However, a number of recent in vitro and in vivo studies have demonstrated that the climbing fiber-elicited calcium response is graded, and that variations in the amplitude of the calcium response can modify the probability that associative plasticity is induced. The current manuscript extends this work by identifying inhibition from the molecular layer interneurons (MLIs) as a signal that can regulate the calcium response elicited by the climbing fibers. The authors employ a powerful combination of complementary in vivo and in vitro calcium imaging, optogenetic and pharmacogenetic manipulation, and slice physiology. In response to the previous review, the authors have conducted a number of additional analyses, which increase the rigor and strengthen the conclusions. In addition, they included new data from experiments imaging the parallel fiber axons during their cued licking task. Overall, this study represents a significant advance in understanding how the cerebellum implements learning, in particular, the events governing the induction of associative LTD at the parallel fiber-Purkinje cell synapse. The reviewers did not think additional experiments were necessary for this to make a valuable contribution, however, they felt that some additional revisions of the text were needed to better acknowledge and discuss some of the limitations of the current study that were raised in the previous review, and which were not fully addressed by the additional analyses and experiments provided in the revised manuscript.Essential revisions:1) The manuscript provides convergent evidence that inhibition from the molecular layer interneurons (MLIs) can gate supralinear calcium responses to combined parallel fiber and climbing fiber activity. This is likely correct. However, it is also difficult to rule out the possibility that in vivo, the chemogenetic suppression of MLIs could have resulted in changes (increases) in granule cell/parallel activity, which could contribute to the enhanced calcium responses. The authors need to be more forthcoming and explicit in the manuscript about acknowledging this possibility and the limited extent to which their evidence addresses it.Their argument that argument that parallel fiber activity is not altered by suppression manipulation of MLI activity hinges on:a) new data showing that in the absence of MLI manipulation, calcium imaging-based measurement of parallel fiber activity closely tracks MLI activity  and an assertion in their point-by-point response that "unilateral chemogenetic disinhibition of MLIs in Crus II did not affect the population response of MLIs (data not shown). There are some concerns with this argument. First, just because the activity of two populations of neurons is similar under one measurement condition, it does not mean that it will be similar under all conditions, such as experimental manipulation of inhibition. Second, the whole point of the chemogenetic manipulation of MLIs is to suppress their activity, so why does it not affect the population response? Are we missing something? Third, the Kit promoter has some expression in Golgi cells, which directly inhibit the granule cells-this needs to be acknowledged.b) Lack of change in the behavior. The additional data provided in Figure 2—figure supplement 3 is helpful, but does not fully address the possibility of other, unmeasured behavior differences , which is why the reviewers had asked for sample videos with and without chemogenetic suppression. Also, the lack of behavioral difference does not rule out a difference in the granule cells that does not affect the behavior, but could contribute to calcium transients in the Purkinje cells.In the absence of more direct recordings from the granule cells or parallel fibers comparing responses in the presence or absence of the disinhibition manipulation, we would be satisfied with a more thorough acknowledgement and discussion of the above caveats in the manuscript.2) Figure 1—figure supplement 1B and 1E show that PF activity is well correlated with licking behavior after the cue. However, it is not very clear in Figure 1—figure supplement 1D. In particular, fluorescence signals begin to increase mostly before licking starts in the B3 bouton. It is therefore important to show PF and MLI activity not only when the cue induces licking but also fails to induce licking as described above. In addition, please indicate in Figure 1—figure supplement 1D when the cue was presented. Otherwise, readers cannot tell which licking bouts are learned behavior.3) In the first round of review, one of the reviewers asked more discussion of the cued licking task, for example, which components of the lick task are cerebellum dependent-acquisition of learning. The authors address the comment, but their rebuttal letter has addressed the comment better than the manuscript itself. It is important to let readers know that it is currently unclear (1) the role of climbing fibers in the cued licking task, and (2) which aspects of this learning task are regulated by the cerebellum. These limitations do not diminish the value of this study. Essential revisions:1) Because long trains of PF stimulation can induce a supralinear PF+CF effect on calcium, the authors should rule out the possibility that pharmo- and opto-genetic perturbations drives an increase in PF activity . The most direct way to achieve this would be to perform calcium imaging in granule cells and check that activity is similar between treatment and sham conditions. It may also be helpful to provide a more thorough analysis of the effects of the MLI manipulations on behavior. Is enhanced calcium still observed with CNO if the analysis is limited to CNO and control trials with the behavior as closely matched as possible ?2+ imaging approach. Thus, by inference, disinhibition did not affect the overall level of granule cell activity. This rules out the possibility that enhanced Ca2+ signaling in PCs was the result of a CNO-induced change in the presynaptic activity level of granule cells.We have collected a new set of data using GCaMP6f to measure the activity of parallel fiber axons during our cued-licking task. Parallel fibers were robustly activated during the consumption of water and were relatively inactive in the absence of licking . Parallel fiber activity also closely tracked licking kinematics. Interestingly, the movement-induced activation of MLIs was essentially an exact match of that of the average response of the parallel fibers in the same region. This indicates that MLI activity measurements can be used to infer the overall activity of the granule cell population. Unilateral chemogenetic disinhibition of MLIs in Crus II did not affect the population response of MLIs, measured using our Ca2+ activity. These points are now emphasized in the revision.Addressing the specific question of parallel fiber signaling onto Purkinje cell dendrites with and without MLI block is not technically feasible in awake behaving animals at this time. We are therefore left with relying on the reduced preparation experiments where we show that shifting the balance between parallel fiber excitation and MLI inhibition of PCs can likewise affect PC Ca2+ activity in a subset of mice showing the smallest deviations in lick rate with chemogenetic disinhibition, compared to control. MLI activity suppression continued to enhance climbing fiber-evoked Ca2+ events in PCs from these mice . Moreover, limited behavioral differences indicate that granule cell activity (which tracks behavior) is also unlikely to change with chemogenetic inhibition of MLIs, which further supports our conclusion that the observed alteration of PC Ca2+ signaling by chemogenetics is not due to activity changes in granule cells.We apologize for not being forward in our reporting of potential motor changes with chemogenetic suppression of MLI activity. We actually went out of our way to identify conditions that were not conducive to affect behavior as this could complicate the interpretation of our results. We now present our analysis showing the absence of changes in average licking performance following unilateral suppression of MLI activity in the small area of left Crus II under our imaging window . We do not think this result contradicts our previous work where we reported a small, but significant reduction in lick rate with MLI activity suppression by hM4d activation . This is because in this prior work, we inhibited MLI activity bilaterally in large swaths of both left and right Crus II, a much more robust perturbation of the MLI population controlling orofacial function. At the reviewers’ request, we examined PC Ca2) More thorough analysis of the data, and more complete presentation of the results are needed throughout the manuscript.We have made extensive new additions to the manuscript including many new supplemental figures to address each of the reviewers’ concerns.a) In the analysis of the in vivo calcium events, the authors focus on the unitary calcium responses, and exclude the multipeaked responses from most pf the analyses. The description of how the unitary events were identified is scanty . The authors should provide more information about how they did the analysis and provide an analysis of the robustness of the results to their specific choices for the analysis. What does the distribution of all (single and multipeaked) calcium event amplitudes look like? What is the distribution of single, double, triple peaked events? Does the latter differ during cued licking behavior vs. spontaneous? The example trace in Figure 1C suggests there is surprisingly large variation in the size of unitary events (a ~3-fold difference in the peak with the green check mark vs. the first peak in the multiple event)-this makes me wonder how many of the unitary events selected for analysis are actually composed of two smaller calcium events occurring close together in time. What is the temporal resolution for discriminating one vs. two closely spaced events? A more careful consideration of how the choice of analysis could affect the results and the interpretation of the results is needed.2+ signals. Specifically, we used a previously published inference algorithm  to identify all climbing fiber-evoked Ca2+ events in PCs, whether or not they occurred during the decay of preceding events. The reviewers refer to these overlapping events as “multi-peaked”. We are hesitant to use this terminology as it seems to imply some sort of physiological relevance rather than simply reflecting that climbing fiber-evoked events can occur in succession and the decay time of GCaMP6f is relatively slow . We rejected overlapping dendritic Ca2+ events from our initial analysis because of possible uncertainties of GCaMP6f nonlinearity. Therefore, our criteria for inclusion was that an event occurred at least 500 ms from proceeding or following events, a window that allows for GCaMP6f fluorescence to decay back to baseline levels . We have now included a better description of our selection criteria and rationale in the Materials and methods section.We apologize for the inadequacy of our description of unitary event selection for analysis of PC dendritic Ca2+ event rate in PCs is low, about 1.5 Hz. We found that these distributions don’t change during motor behavior .In response to the reviewers urging, we now report the distribution of single vs. overlapping events. Isolated, single events comprise the majority of the response population; this makes sense because the average Ca2+ event amplitudes increased during movement. This is a robust result. New analysis shows that this holds true not only for discrete single events, but also for all other events including overlapping responses . Regarding the discrimination of overlapping Ca2+ events, our imaging rate is 30 Hz (~33 ms); this defines a theoretical temporal resolution by which we could distinguish two closely spaced dendritic events. That said, we don’t believe that unresolved, closely-spaced responses appearing as single events accounted for the difference in response amplitude with chemogenetic disinhibition. If so, this would have been reflected in the presynaptic activity of climbing fibers. Because Ca2+ event amplitudes (and frequency) did not change in climbing fibers with molecular layer disinhibition, this argues strongly against this possibility. We now state this in the text.A major finding of our report is that with chemogenetic suppression of MLI activity, Ca2+ events. Identifying the mechanistic underpinnings of this variability is a major goal of ours. That similar variability is apparent in the amplitudes of Ca2+ events in climbing fibers indicates the PC dendrites are simply integrating the level of presynaptic activity from these inputs. We hypothesize that MLI-mediated gating of dendritic nonlinearities allow for the faithful conversion of presynaptic climbing fiber activity into postsynaptic Ca2+ signals in PCs. However, this investigation is beyond the scope of the current report.Like the reviewers, we are fascinated by the variability in the amplitudes of PC dendritic Ca2+ signaling after MLI disinhibition. If I understand correctly the figure also indicates that the other ~37% has a substantial decrease of the Ca2+. This should be addressed, also in relation to the chance of random changes in Ca2+ signaling . Similarly, the remark about the 'arbor-wide' influence should be re-worded to more careful terms.b) The authors show in Figure 3C that ~63% of the dendritic area shows enhanced Ca2+ activity across all pixels of identified PC dendrites . This analysis indicates a widespread effect . In addition, we have re-worded the Results section, taking caution about the use of the phrase ‘arbor-wide influence’.To address this concern, we have changed our analysis approach and now include use of receiver operating characteristic (ROC) curves to diagnose the probability of disinhibition-induced shifts in Cac) For Figure 5D and Figure 6, it would be very helpful if the evoked PF and CF transients were also shown individually, before the summed transient is shown. Also, please state clearly if the summed response for the suppressed inhibition is based on the PF with or without inhibition.2+ transients in Figure 5D . This should help readers understand our approach for quantifying supralinearity in dendritic PC Ca2+ signaling more easily. The same method applied for dendritic Ca2+ activity measurements in Figure 6. We now state, in the accompanying legend of Figure 5, that the summed response for the suppressed condition is based on a PF stimulus without feed-forward inhibition .We now show both the individual, parallel fiber- and climbing fiber-evoked Cad) Figure 6G and associated text suggest that the MLIs exert a graded effect on the climbing fiber-elicited calcium response. Is there truly a graded effect on amplitude, or might it be an effect on the probability of an additive vs. supralinear event of a more uniform amplitude? Additional analysis could distinguish these two equally interesting possibilities.2+ transients could distinguish these possibilities. However, the feasibility of such analysis has not been established for PCs, at least to our knowledge. This is because single trial analysis is made difficult due of the low signal-to-noise level of climbing fiber-evoked responses. We could change our recording parameters in an attempt to help improve the quality of evoked Ca2+ signals . This would be very laborious and time consuming. In the end, parsing which mechanism is responsible for the observed effect would not change the overall interpretation of our results as both possibilities are interesting and relevant. Therefore, we have removed the term “graded” from the manuscript in favor of simply stating that the effect of parallel fiber excitation on climbing fiber-evoked Ca2+ signaling in PCs was dependent on the level of MLI activity.Single-trial analysis of climbing fiber-evoked Cae) The point of Figure 1 is that the amplitude of the calcium events is the same during movement or no movement. However, in Figure 2F, this does not seem to be the case for the hM4d animals, even in the absence of CNO. If the calcium sensing is nonlinear, this could potentially influence what is measured with CNO. This possibility should be considered.2+ response with movement, compared to events in the absence of movement, in hM4D-expressing mice. However this difference is not significant. We now state this in legend of Figure 2. The major concern with uncertainties regarding GCaMP6f nonlinearity is whether there is a faithful reporting of the relative change in amplitudes (including the absence of change due to saturation). This is compounded for responses that occur in the decay of others because Ca2+ unbinding isn’t yet complete. This is a major reason why we avoided including such events in our initial analysis . Heeding this concern, we were also hesitant about making significant conclusions based on how much change we observed in the disinhibited condition. Thus, we don’t believe that enhancement of Ca2+ signaling by GCaMP6f can be explained by a nonlinearity, although the absence of effect may warrant caution. In response to the reviewers, we now state in the text that GCaMP6f is known to be nonlinear but, based on previous reports, is sensitive enough to report small changes in climbing fiber-evoked PC Ca2+ signaling to different behavioral stimuli. This point is quite moot later in the manuscript when we show that enhanced Ca2+ signaling can be uncovered with chemogenetic-induced, molecular layer disinhibition.The reviewers point to a trend towards a larger Caf) There are some places where individual examples are shown, but no group data, for example, Figure 4E.We have changed Figure 4E to include group data; we can find no other instances where group data are not reported either in the text or in a separate Figure panel.g) There are some places where results are presented without statistics. For example, is there is a significant difference between movement vs. no movement and between CNO vs. control in Figure 3FG? Also, the error bars on the CNO-control PC data for simultaneous PC/MLI imaging  are very large. Please provide the same statistical analyses for these data as were used for the data in Figure 2F to justify the claim that the difference is positive. Also, please provide the same analysis for the climbing fiber imaging data in Figure 4G, where the difference appears to be negative, but is asserted to not differ from zero.2+ activity was significantly different following chemogenetic suppression of MLIs). We apologize for not pointing out this figure panel in our original submission. This has now been corrected.The statistical comparisons for the data shown in Figure 3F and 3G are presented in Figure 3H . The difference trace, like those shown in Figure 2C-2E, are for the reader’s convenience. The statistical test for this comparison is reported in the text; it was non-significant .For activity measurements in climbing fibers, we compared the peak amplitudes of averaged Ca2+ activity during cued licking, we show the subtracted difference between responses measured in control and with molecular layer disinhibition . We chose to present the subtracted difference because it facilitated comparison to the activity plot of MLIs . This was also for the reader’s convenience but, as requested, we performed a statistical analysis of this dataset . Based on the reviewers’ comments, we believe presenting the data as we did may have caused confusion. Therefore, we now show responses of trial-average PC activity in the two conditions used to generate the difference plot (control and with chemogenetic MLI activity suppression) as well as an accompanying statistical analysis of these data .Regarding our results examining trial-averaged PC Cah) To support the claim that peak PC ΔF/F does not co-vary with MLI activity , please plot compute the correlation between the peak amplitude and MLI activity for all dendrites and complex spike event, and display a scatterplot of these values.2+ events in PCs and the corresponding level of MLI activation. However, in response to the reviewers, we have provided scatterplots from three representative mice showing all PC dendritic events and the corresponding level of activity in surrounding MLIs .No relationship was found.As shown in our analysis of mean activity , there is no relationship between the amplitude of climbing fiber-evoked dendritic Cai) A video of mouse behavior and two-color imaging  would be useful.2+ activity measured simultaneously in PCs and MLIs using a dual-color, Ca2+ indicator imaging approach. The average responses are demarcated based on their correspondence to cell type and aligned to the onset of behavior (licking). We are not sure how the addition of a video would help clarify our approach or provide any additional insight into this result to readers.We show a plot of Caj) Figure 5D shows that MLI inactivation induces a supralinear interaction between PF and CF stimulation on dendritic calcium. Is the same true of the somatic membrane potential? Please overlay plots of the PF+CF and PF+CF (sum) for control and light conditions  in Figure 5F, and make a similar plot for the ephys data in the long pulse train experiments .2+ signaling . This is likely mediated through the passive spread of voltage into the dendritic compartment, preventing parallel fiber-mediated inactivation of Kv4 currents . It follows then that GABAAR-mediated hyperpolarization, elicited by MLI activity, is sufficient to prevent the supralinear interaction of parallel fibers and climbing fibers in PC dendrites, as observed in our experiments. At the reviewers’ request, traces of complex spikes with and without preceding parallel fiber stimulation are shown superimposed in Figure 5F for both control and the responses with optogenetic suppression of MLIs. For the data presented in Figure 6, these are included as a supplement additional figure .Hyperpolarization of the membrane potential by somatic current injection diminishes climbing fiber-evoked responses in PC dendrites including supralinear Cak) Was there any correlation between calcium transient amplitude and behavior? For instance, were transients larger on trials with longer or mistimed licking bouts? Similarly, did the correlation between PC calcium and MLI calcium change with any aspect of behavior? Although the authors have addressed some of these questions in previous work, it would be helpful to have this information for the current manipulations and data.2+ responses. To address this point carefully, will require more exacting control of the movement task as well as additional tools to measure and manipulate neural activity in the granule cell and molecular layers. Thus, this is beyond the scope of the current study. As the reviewers point out, we have previously reported that the rate of climbing fiber-evoked Ca2+ events in PC dendrites increases at the onset of licking while MLI activity increases and decreases with changes in licking rate during water consumption. This is now discussed in greater detail.We did not observe significant changes to average licking behavior with unilateral chemogenetic suppression of MLI activity in left Crus II. This is now reported in the manuscript . A more in depth examination of deviant licking, perhaps reflecting motor errors that lead to learning, was not pursued. Regarding motor errors and climbing fiber-evoked signals, it is certainly a future interest of ours to understand the behavioral conditions conducive to producing PC supralinear Cal) In general, the figure legends and text describing the figures should be edited to be more precise and make it easier for the reader to understand what is in the figure. For example, in Figure 2G and associated text, I am guessing that the "totality of all calcium responses" includes the multipeaked as well as the unitary responses, but after considerable effort, I am still not sure. Another example, it is not clear whether the averaged calcium activity in Figure 1F is from one mouse or all mice. Please carefully review all figure legends to make sure that the reader is provided with sufficient information about what is in each figure panel.2+ activity (including overlapping events spaced closely in time) in all identified PCs across all trials. We have attempted to improve the clarity of our language regarding this dataset and have added a new figure to illustrate this result more directly . We now report numbers for Figure 1F in the legend.The “totality of all calcium responses” was meant to indicate that we simply averaged all Cam) Please provide time units for the event probability plots to allow comparison across figures, and with previously published climbing fiber firing rates. I would expect the probabilities in Figure 1E and Figure 4E to be similar, but they differ by twofold. How does this compare with the typical climbing fiber rate of 1 Hz?2+ events is the same for both climbing fibers and PCs. The increase in peak Ca2+ event rates at the initiation of licking is also not significant between climbing fibers and PCs (there is large amount of variability in the climbing fiber response); we don’t report this because our preference is to more carefully compare climbing fibers and PCs using simultaneous dual-color imaging of pre- and post-synaptic activity in the same mouse which we hope to publish in the near future. The rates of Ca2+ events detected in PCs in our experiments closely matches that of previously published values. This is now stated in the text with accompanying references. To our knowledge, we are unaware of anyone else who has measured activity rates directly from climbing fibers, so we are proud to say that this is a first!We changed the plots to include time units (rate/frequency). We show that the average rate of Ca3) The current results would have more impact if more effectively framed in the context of what is already known about variations in climbing fiber-triggered responses in the Purkinje cells, how the current work extends what is known, the conclusions that can be drawn, and any caveats.We thank the reviewers for highlighting areas of our manuscript where we could include clarification to increase the impact of our work.2+ signaling" in the Title and last sentence of the Abstract and "circuit modifications" in the first sentence of the Abstract), given that plasticity is not tested in thea) Previous work has associated the calcium transients with plasticity, however, the reviewers thought there was too much emphasis on plasticity  Najafi et al. have reported that climbing fiber-associated calcium responses are enhanced when they occur during a learning task as compared with the spontaneous calcium events, which are presumably due to spontaneous climbing fiber spiking. In contrast, the current results indicate that the calcium responses during the well-learned lick task are the same as the spontaneous calcium events. The authors cite Najafi, but don't directly compare the two results so that the reader can effectively appreciate the new finding that the calcium responses are not always enhanced in the behavioral context. This would better frame the current Results section than the current statement in the Introduction "whether CF Ca in vivo is unclear" and would explain why the results in Figure 1D are described as "Surprising".2+ signaling in PCs. A detailed comparison of the work by Najafi et al., contrasting their findings to ours, is also provided.We thank the reviewers for pointing this out. We now emphasize the novelty of our findings regarding that lack of behavior-related augmentation of Ca2+ signal in Purkinje cells during the movement in the lick task is needed. From the example mouse data in Figure 1B, it is difficult to tell how much of the behavioral response and neural activity is learned (a conditioned response) versus an unconditioned response to the water — it looks like the majority of the neural and behavioral response could be unconditioned. Which components of the lick task are cerebellum dependent-acquisition of learning? expression of the condition response? performance of unconditioned licks? Discussion of these issues would help to clarify the potential role of the calicum transients in the behavior.c) More discussion of the cued licking task, and the role of the Ca2+ signals in guiding learning. But we conjecture that learning is not apt to be occurring during this well-practiced motor behavior. That said, climbing fiber activity has been shown to be important for motor memory retention . It may be that the climbing fiber-evoked Ca2+ signals that we studied are import for maintenance of some earlier aspect of behavioral adaptation during task training . Certainly, there is an abundant literature indicating that complex spikes evoked by climbing fibers are important for online motor control. As discussed, MLI-mediated suppression of nonlinear Ca2+ signaling could allow climbing fibers to multitask. Supporting both online motor control through the influence of complex spike bursts at the PC soma separate from the acquisition of plasticity in the dendrite.We have added to the Discussion section to address some of these points. In summary though, we have not yet identified which aspects of lick-related behavioral refinement are due to this region of the cerebellum nor do we understand the importance of the climbing fiber-evoked responses in PCs, elicited at licking onset, to task performance. It is enticing to speculate on the potential role of these PC Cad) In Figure 5 and Figure 6, might the lack of supralinear calcium responses result from the use of a suboptimal parallel fiber-climbing fiber pairing interval?2+ responses were not apparent with feed-forward inhibition intact. These new data are now included in the manuscript .We tried different pairing intervals. However, supralinear Ca[…] Essential revisions:1) The manuscript provides convergent evidence that inhibition from the molecular layer interneurons (MLIs) can gate supralinear calcium responses to combined parallel fiber and climbing fiber activity. This is likely correct. However, it is also difficult to rule out the possibility that in vivo, the chemogenetic suppression of MLIs could have resulted in changes (increases) in granule cell/parallel activity, which could contribute to the enhanced calcium responses. The authors need to be more forthcoming and explicit in the manuscript about acknowledging this possibility and the limited extent to which their evidence addresses it.In the revised manuscript, we now attempt to make these points more explicit and state the limitations of our approach/conclusions.Their argument that argument that parallel fiber activity is not altered by suppression manipulation of MLI activity hinges on:a) new data showing that in the absence of MLI manipulation, calcium imaging-based measurement of parallel fiber activity closely tracks MLI activity  and an assertion in their point-by-point response that "unilateral chemogenetic disinhibition of MLIs in Crus II did not affect the population response of MLIs (data not shown). There are some concerns with this argument. First, just because the activity of two populations of neurons is similar under one measurement condition, it does not mean that it will be similar under all conditions, such as experimental manipulation of inhibition. Second, the whole point of the chemogenetic manipulation of MLIs is to suppress their activity, so why does it not affect the population response? Are we missing something? Third, the Kit promoter has some expression in Golgi cells, which directly inhibit the granule cells-this needs to be acknowledged.Kit::Cre driver line.We now state/discuss these caveats. The chemogenetic inhibitor hM4d is thought to work by a presynaptic action . This would block release of GABA from MLIs, but not necessarily affect the calcium activity driven in large part by parallel fiber input. The revision includes specific mention of possible Golgi cell expression of hM4d in our b) Lack of change in the behavior. The additional data provided in Figure 2—figure supplement 3 is helpful, but does not fully address the possibility of other, unmeasured behavior differences , which is why the reviewers had asked for sample videos with and without chemogenetic suppression. Also, the lack of behavioral difference does not rule out a difference in the granule cells that does not affect the behavior, but could contribute to calcium transients in the Purkinje cells.In the absence of more direct recordings from the granule cells or parallel fibers comparing responses in the presence or absence of the disinhibition manipulation, we would be satisfied with a more thorough acknowledgement and discussion of the above caveats in the manuscript.We now point this out in the Results section. Specifically we state that we cannot rule out behavioral alterations such as those described by the reviewer.2) Figure 1—figure supplement 1B and 1E show that PF activity is well-correlated with licking behavior after the cue. However, it is not very clear in Figure 1—figure supplement 1D. In particular, fluorescence signals begin to increase mostly before licking starts in the B3 bouton. It is therefore important to show PF and MLI activity not only when the cue induces licking but also fails to induce licking as described above. In addition, please indicate in Figure 1—figure supplement 1D when the cue was presented. Otherwise, readers cannot tell which licking bouts are learned behavior.The data for PF activity have been added to Figure 1—figure supplement 1. It is essentially a flat line. The result for MLIs is the same, but has been excluded from the plot for clarity. This is stated in the figure legend. The timing of the sound cue is now shown in panel D.3) In the first round of review, one of the reviewers asked more discussion of the cued licking task, for example, which components of the lick task are cerebellum dependent-acquisition of learning. The authors address the comment, but their rebuttal letter has addressed the comment better than the manuscript itself. It is important to let readers know that it is currently unclear (1) the role of climbing fibers in the cued licking task, and (2) which aspects of this learning task are regulated by the cerebellum. These limitations do not diminish the value of this study.Provided that the reviewers found our response in the rebuttal informative, we simply moved these sentences into the Discussion section of the revision.


Children eat less than recommended amounts of vegetables. Repeated taste exposure can increase children’s acceptance of initially disliked vegetables. However, implementation of this strategy is lacking. We conducted a pilot study to assess the feasibility of implementing an evidence-based intervention to promote liking of initially disliked vegetables among children enrolled in a YMCA summer camp.We adapted a research-tested intervention to promote child liking of vegetables for implementation in small groups. In summer 2015, 50 children aged 7 to 12 years were invited to taste 5 initially disliked vegetables daily for 10 days.Children rated how much they liked vegetables on a 5-point emoji-like faces Likert scale at baseline and 2- and 4-week follow-up. The mean ratings for liked and initially disliked vegetables were estimated over time using mixed effects modeling.P = .003) for the 5 targeted vegetables but not for the other nontargeted vegetables, as predicted.We achieved excellent participation of parents and children; however, we experienced nonstudy-related attrition caused by disenrollment of some children from the weekly camp program. The average liking increased over time (linear trend, This pilot study suggests that repeated vegetable tasting opportunities offered by community programs may be a practical strategy for introducing low-income, young children to new or initially disliked vegetables. The study demonstrates the feasibility of implementing a health promotion strategy that has the potential to improve population health in a community setting in an underresourced neighborhood. Most children eat less than the federally recommended daily amount of vegetables . GettingHaving children eat more minimally processed fruits and vegetables leads to reduced consumption of energy-dense foods in families , althougSouth Los Angeles is part of Service Planning Area (SPA) 6 in Los Angeles County, where residents are mostly African American (28.5%) or Latino (67.7%). Of the 8 SPAs in Los Angeles County, SPA 6 has the largest proportion of adults with less than a high school education (41.6%) and a household income of less than 100% of the federal poverty guidelines (33.6%). It has the lowest number of adults who consume 5 or more servings of fruits and vegetables a day (9.6%) and the highest proportion of adults who are classified as obese (34.1%) .To promote healthier eating, we adapted a research protocol from a trial originally conducted in British kindergartens and elementary schools  called TThe Tiny Tastes program repeatedly exposes children to small bites of initially disliked vegetables to increase their liking for these vegetables. Our pilot study objectives were to 1) assess the feasibility of adapting the evidence-based intervention and implementing it in the busy environment of a YMCA summer camp, and 2) explore the short-term impact of the program on children’s liking of initially disliked vegetables and on their willingness to try new foods.In North America, the YMCA is primarily a community sports facility, open to all, regardless of religion, social class, sex, or age . The 2,7Participants were recruited from the Weingart YMCA Wellness and Aquatic Center in South Los Angeles from a pool of 109 children aged 7 to 12 years who were enrolled in the summer day camp. YMCA summer day camps provide a safe and enriching environment for children to participate in various healthy, developmentally appropriate activities and learning experiences when school is not in session. Generally, campers are divided into smaller groups to participate in structured activities, including sports, and receive morning and afternoon snacks and lunch.Bilingual research staff passed out information about the study in English and Spanish to parents who dropped off their children in the morning, including a form that parents could mark to opt their child(ren) out of the study (passive consent). All children spoke English. At scheduled times, the camp director brought small groups of age-eligible, study-eligible children to the test kitchen where research staff explained the study and asked if children wanted to participate. The study was approved by the University of California, Los Angeles (UCLA), Institutional Review Board.P < .001). The Tiny Tastes program . Follow-up assessments were conducted immediately after the last exposures (2-week follow-up) and after 14 additional days of nonexposure to assess short-term maintenance (4-week follow-up). Research staff were trained on the study protocol and worked side-by-side with the investigators during the implementation phase. Participants received $10 for each completed assessment, up to $30 total per participant.For implementation of the program during the YMCA summer day camp, the study was renamed Eating Veggies is Fun!, because it translates well into Spanish, ¡Comer Vegetales es Divertido! Eligibility was expanded to include children aged 7 to 12 years. We aimed to enroll 50 children in a prospective cohort study with no control group. Sample size was constrained by the resources we had and by the number of children enrolled in this community venue at the time of the study. Research staff conducted the intervention and assessments in small group sessions (5 to 15 children), instead of the individual sessions used in Tiny Tastes. The baseline assessment included a 5-point emoji-like faces scale instead of Tiny Tastes’ 3-point scale with the categories Super yuck, yuck, okay, yum, super yum for 12 vegetables and 8 fruits that are commonly consumed in California and readily available in grocery stores . As in tWe assessed demographic characteristics  at baseline. At baseline and follow-up interviews, we assessed availability of fruits and vegetables at the child’s home , consumption of fruits and vegetables yesterday  and liking of 12 vegetables and 8 fruits on a 5 point-Likert scale with faces (1 = super yuck to 5 = super yum). The fruit and vegetable assessment Likert scale was based on the research conducted by Birch and Sullivan  and has t tests for continuous variables and χ2 tests for categorical variables. We compared liking of vegetables at baseline and follow-up using paired sample t tests for continuous variables, McNemar tests for dichotomous variables, and Wilcoxon signed rank tests for ordinal variables. The mean ratings for liked and initially disliked vegetables were also estimated over time using mixed effects modeling, including sex, age, and race as covariates. Analyses were conducted using IBM SPSS Statistics  and Stata .We compared demographic characteristics and liking of vegetables at baseline between study completers and noncompleters using 2-sample Over 6 weeks , we enrolled 50 children. Only 2 children from 1 family had a parent who opted them out of the study, and all children who attended the orientation agreed to participate. Although we did not record how much was consumed by each child, we did observe that all children who were enrolled in the study participated in the tasting. After repeated exposure to the 5 target vegetables, 38 children completed the 2-week follow-up assessment (76% retention). After an additional 2-week period following cessation of the vegetable tastings, 28 children completed the 4-week follow-up assessment (56% retention). More than 80% of study attrition was due to children’s absence on the day of assessment, usually because their participation in the summer camp program had ended.Study participants were 26 boys and 24 girls, 58% were African American, 24% were Latino, and 18% reported mixed racial/ethnic backgrounds . Their m2(1) = 8.66, P = .003). The 7 vegetables that participants already liked at baseline included broccoli, carrots, celery, cucumbers, spinach, and tomatoes. There was no significant linear trend for the adjusted mean liking for the 7 nontargeted vegetables. The means were 4.0 at baseline, 3.8 at 2-week follow-up, and 3.8 at 4-week follow-up.A mixed effects modeling analysis that including sex, age, and race as covariates showed a significant increase in the liking for the 5 targeted vegetables . The adjP = .02). The liking for other vegetables not targeted in the study remained unchanged, except for a borderline significant drop in liking for the nontargeted vegetable celery (P = .05). There were no significant changes in how much children reported liking to try new foods and in the child’s reported accessibility to and consumption of fruits and vegetables “yesterday” (When examined singly, only the increase in liking for jicama was significant (sterday” . In analThe modified research protocol was easy to integrate into the daily schedule of a YMCA summer day camp and the study protocol was acceptable to parents and children, as evidenced by the excellent participation rate among children who remained enrolled in the summer program. Our sample was predominantly African American, which may explain why many children were not familiar with jicama, a vegetable that is commonly eaten by Latinos. Children unfamiliar with jicama did not provide a baseline rating, thereby further reducing the sample size for this vegetable for the baseline and 2-week follow-up comparison. Despite the limited sample size, we were able to show significant increased liking for jicama among children who did provide a baseline rating, which was maintained at 4-week follow-up. Jicama was the top rated vegetable at both follow-up assessments, which suggests that repeated tasting can also increase the liking of specific unfamiliar foods. This conclusion is consistent with findings of a study by Wardle and colleagues .The fact that the liking increased only for vegetables to which children were repeatedly exposed, but not for the other vegetables to which the children were not repeatedly exposed, is consistent with the hypothesis that repeated taste exposures will increase children’s liking of initially disliked vegetables. Our finding is consistent with literature showing that repeated exposure leads to increased liking for the target foods ,16,23,24Repeated exposure to the 5 vegetables did not translate into participants reporting an overall increased willingness to try other new foods. Continued broadening of the children’s repertoire of acceptable vegetables would require their sustained, repeated exposure to a wider variety of unfamiliar vegetables.Our study was limited to a single YMCA site, which limits generalizability. Other limitations include use of a single group pretest–posttest comparison without a control group and a small sample size. The study was implemented during a summer day camp, in which children enrolled for a week at a time. Many children did not attend the YMCA camp for the whole summer, which caused substantial study attrition at the 4-week follow-up time point and prevented a longer term follow-up assessment. The program was implemented with small groups of children rather than with individual children as in the Tiny Tastes protocol, which would not be feasible in a day camp setting. We acknowledge that a strong positive or negative reaction to a vegetable from a participant could have influenced other children in the group. Our research staff tried to keep children busy during the tasting sessions to reduce peer influence on liking, similar to how children were tested in other studies . A populIn this pilot implementation study, research staff worked with small groups of children to facilitate consistent implementation of the vegetable tasting and to administer the assessments. However, in a future implementation, camp staff could incorporate the tasting for all campers during snack time, especially if there is no need to administer assessments. Thus, vegetable tasting could be incorporated with minimal disruption to other activities and the cost of vegetables could be included in the camp food budget. We recommend that summer YMCA programs add the goal of promoting a healthy eating pattern that includes eating more vegetables into their mission statement.This pilot study suggests that repeated vegetable tasting opportunities offered by community programs may be a practical strategy for introducing low-income, young children to new or initially disliked vegetables. The study demonstrates the feasibility of implementing a health promotion strategy that has the potential to improve population health in a community setting in an underresourced neighborhood. Given the continuing failure of traditional weight-control strategies to halt the increase in pediatric obesity  and give